LECTURE NOTES

ON

ENZYMOLOGY FOR
POSTGRADUATE STUDENTS

BY

B.S. FAGBOHUNKA
Ph. D (Enzymology)

1.0

Introduction

2.0

Objectives

3.0

Enzymes

3.1

Nature & Properties of enzymes

3.2

Co-enzymes

3.3

Classification of enzymes

3.4

Enzyme kinetics

3.5

Lineweaver-Burke Plot

3.6

Enzyme inhibition

4.0

Conclusion

5.0

Summary

6.0

Tutor Marked Assignment

7.0

Further Reading and other Resources

CLINICAL ENZYMOLOGY

1.0

INTRODUCTION
Enzymes are biological catalysts. Almost all the reactions that take place in living cells are
catalysed by enzymes. Although there are ribozymes i.e. catalytic RNAs, most enzymes are
proteins. In this unit you will learn about the nature of enzymes and enzyme kinetics and
inhibition.

2.0

OBJECTIVES
At the end of this suit unit you are expected to know the following:
The nature of enzymes.
Classification and nomenclature of enzymes.
Coenzymes.
Enzymes Kinetics.
Enzyme Inhibition.

3.0

ENZYMES: BASICS
Most enzymes are proteins. This means that proteins do not have the monopoly to
function as enzymes. Actually, there are catalytic RNAs or ribozymes. However, without
the presence of a non-protein component called a cofactor, many enzyme proteins lack
catalytic activity. The enzyme without the cofactor is called an apoenzyme. The enzyme
and cofactor is called the holoenzyme. When the cofactor is an organic molecule, it is
called a coenzyme. When a cofactor is bound so tightly that is difficult to remove without
damaging the enzyme, it is called a prosthetic group.

3.1

Nature and Properties of Enzymes

Enzymes (E) increase the rate of chemical reactions taking place within living cells
without themselves suffering any overall change. The reactants of enzyme-catalyzed
reactions are termed substrates (S). Each enzyme is quite specific in character, acting on a
particular substrate or substrates to produce a particular product (P) or products.
They increase the rate of forward and reverse reactions by the same factor. Therefore, they
do not change the direction of reactions, nor do they change the equilibrium ratio between
products and reactants.
Since most enzymes are protein, they also have the properties of protein; particularly, they
are thermolabile. The optimum temperature for most enzymes is about 40 oC. Above the
optimal temperature, the enzyme is denatured and so stops to act. The activity of enzyme
is also dependent on the pH of the medium. Some require neutral, some acidic and others
alkaline medium.
Activities of enzymes can be influenced by agents known as modulators. Modulators that

increase the activities of enzymes are known as positive modulator or activators while
modulators that decrease the activities of enzymes are known as negative modulators or
inhibitors.
The activity of an enzyme (E) is dependent on substrate (S) concentration. A plot of the
activity of enzyme against substrate concentrate gives a hyperbola.
3.2

Coenzymes
Coenzymes are organic non-protein compounds required by many enzymes for catalytic
activity. They are often water-soluble vitamins, or derivatives of water-soluble vitamins.
Sometimes they can act as catalysts in the absence of enzymes but not so effectively as in
conjugation with an enzyme. Presented below are some co-enzymes, their vitamin
precursors and the reactions in which they participate:
Table 2.1.1 Coenzymes, Vitamin Precursors and Reactions Catalyzed

Coenzyme
1. Nicotinamide

Abbreviation
NAD+

Reaction

Vitamin Precursor

Oxidation-reduction

Niacin/Nicotinic acid

Adeninedinucleotide
2. Flavinadeninedinucleotide
3. Coenzyme- A
4. Adenosinetriphosphate
5. Pyridoxal Phosphate
6. Thiaminepyrophosphate

(B3)
FAD
CoASH

Oxidation-reduction
Acyl group carrier

ATP

Phosphate carrier

Transamination

TPP

Decarboxylation

Riboflavin (B2)
Pantothenic acid (B5)
Pyridoxal (B6)
Thiamine (B1)

7. Biotin

Carboxylation

Biotin

8. Lipoic acid

Hydrogen carrier

Lipoic acid

FH4

Hydrogen carrier

9. Tetrahydrofolate

Cyanocobalamin (B12)

3.3

Classification and Nomenclature of Enzymes

Due to the very rapid development of enzymology and the large number of enzymes, it
became necessary to have a systematic way of naming enzymes. Before, 1961, enzymes
were named based on the reactions that they catalyzed or the substrate. In order to avoid
duplication of the name of enzymes, the International Union of Biochemistry in 1956
appointed a commission- the enzyme commission -to work out a modality for the naming
of enzymes. The enzyme commission gave their report in 1961 and was published in 1965.
The Enzyme Commission (EC) divided enzymes into six (6) main classes on the basis of
the total reaction catalyzed. Each enzyme is assigned a code number consisting of four
elements, usually abbreviated as EC. W.X.Y.Z The first digit shows the class to which the
enzyme belongs as follows:
Oxidoreductases- oxidation/reduction reaction
Transferases- transfer of an atom or group between two molecular
Hydrolases- hydrolysis reactions
Lyases- removal of a group from substrate (not by hydrolysis) usually reacting in the
formation of double band
Isomerases- isomerization reactions
Ligases- the synthesis joining of two molecules couple with the breakdown of
pyrophosphate bond in nucleoside triphosphate
All enzymes, except isozymes have different EC number. Enzymes that catalyze very
similar but non-identical reactions e.g. the hydrolysis of carboxylic acid esters will
have the same first 3 digits, but the fourth digit distinguishes between them by
defining the actual substrate.
Class I:

They catalyze the transfer of H, or O atoms or electrons from one substrate

another. The 2nd digit indicates the donor and the 3 rd digit the acceptor of
the
reducing equivalent (hydrogen or electrons)

Class II:

They catalyze reaction type

AX + B
BX + A
Naming of transferase
ends with X-transferase where X is the
group transferred.

Class III:

Catalyzes hydrolytic reactions of the form A-X+H2O = X-OH + HA

Class IV:

Catalyzes the non-hydrolytic removal of groups from substrates often

leaving double bonds

Class V:

Catalyzes isomerization reactions

Class VI:

Catalyze the synthesis of new bonds, couple to the breakdown of ATP or

another nucleoside triphosphates

X+y+ATP

X-Y+ADP+Pi
Or

X+Y+ATP
3.5

X-Y+AMP+PrPi

Lineweaver-Burk Plot
Because it is easier to work with a linear graph than hyperbola such as we have for the
dependence of enzyme activity against substrate concentration, scientists have evolved
different linear transformations of the Michaelis-Menten equation for the hyperbola. One of
the very popular transformations is the Lineweaver-Burk Plot. This plot was derived by
obtaining the inverse valves for both sides of the Michaelis-Menten equation as follows:
It is easy to calculate the values of K m and Vmax by the use of the Lineweaver Burk Plot
otherwise, known as the double reciprocal plot. To do this, plot the inverse values of the
rate of reaction 1/V against the inverse values of substrate concentration 1/[S].

3.6

Enzyme Inhibition
Inhibitors are substances which tend to decrease the rate of an enzyme catalyzed
reactions. Some combine directly with enzymes while some act on substrates or cofactors.
We shall examine the former. Those inhibitors that bind to an enzymes in a reversible
manner and can be removed by dialysis or dilution to restore full enzymatic activity are
known as reversible inhibitors.
Irreversible inhibitors cannot be removed from enzymes by dialysis or dilution.
Reversible inhibitors usually rapidly form an equilibrium system with an enzyme to show
a definite degree of inhibition which remains constant over a period of time whereas, the
degree of inhibition by irreversible inhibitors may increase over a period of time.
There are seven (7) types of reversible inhibitors:
i) Competitive ii) Uncompetitive iii) Non-Competitive iv) Mixed v) Partial vi) Substrate
and vii) Allosteric Inhibition. In this introductory course, we shall examine the first three
types only.
Competitive Inhibition: Competitive Inhibitors often are substrate analogs i.e. the
closely resemble the substrate and so, compete with the substrate for the binding-site
(active site) of the enzymes. The inhibitor induces no change in Vmax but an increase in
Km.

Fig.2.1.3 Competitive Inhibition

Uncompetitive Inhibitors: bind only to the Enzymes-substrate complex and not the free
enzymes. It alters the Km & Vmax but not the slope.

Fig.2.1.4 Uncompetitive Inhibition

Non-Competitive Inhibition: A non-competitive inhibitor can combine with an enzyme

molecule to produce a dead-end complex, regardless of whether a substrate molecule is
bound or not. Hence, the inhibitor must bind at a different site from the substrate. It binds
to the enzyme and can also bind to ES complex. It alters both the Km & Vmax and the slope
but Km reminds on change.

Fig.2.1.5 Noncompetitive Inhibition

4.0

Conclusion
There are six (6) classes of enzymes that catalyse all the reactions that take place in living
cells in addition to ribozymes.

5.0

Summary
Enzymes are apoenzymes plus cofactors.

Enzymes are classified into 6 classes and their names end with the suffix -ase.
Coenzymes are organic non-protein component of enzymes.
Enzymes Kinetics studies the rate of enzyme catalysis and the associated parameters
e.g. Km and Vmax.
Enzyme Inhibition studies the agents that adversely interfere with catalysis.
6.0

Tutor Marked Assignments

Write short notes on the nature of enzymes.
List the six (6) classes of enzymes and the reactions that they catalyse.
Determine the Km and the Vmax of the enzyme that catalyse the reaction below:

Velocity (mol L-1 min-1)

Substrate Conc.(mol L-1)

147

182

6.67

23
3
1

323

400

20

40

7.0

Further Reading and other Resources

1. Nelson, D. L. and Cox, M. M. (2000) Lehninger Principles of Biochemistry. 3 rd
Edition, Worth Publishers, New York.
2. Stryer, L. (1988) Biochemistry. 3rd Edition, Freeman & Co., New York.
3. Instructors Resource CD-ROM. Lehninger Principles of Biochemistry 3.0
www.worthpublishers.com/lehninger.

Enzymes and their clinical significance

Plasma enzymes can be classified into two major groups. First, a relatively small group of
enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes
zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large
number of enzyme species are released from cells during normal cell turnover. These enzymes
almost always function intracellularly, and have no physiologic use in the plasma. In healthy
individuals, the levels of these enzymes are fairly constant, and represent a steady state in which
the rate of release from damaged cells into the plasma is balanced by an equal rate of removal of
the enzyme protein from the plasma. Increased plasma levels of these enzymes may indicate
tissue damage.
Normal cell turnover Cell necrosis as a result of disease or trauma: Release of enzymes
from normal and diseased or traumatized cells.
Plasma is the fluid, noncellular part of blood. Laboratory assays of enzyme activity most often
use serum, which is obtained by centrifugation of whole blood after it has been allowed to
coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory.
A. Alteration of plasma enzyme levels in disease states
Many diseases that cause tissue damage result in an increased release of intracellular enzymes
into the plasma. The activities of many of these enzymes are routinely determined for diagnostic
purposes in diseases of the heart, liver, skeletal muscle, and other tissues.The level of specific
enzyme activity in the plasma frequently correlates with the extent of tissue damage. Thus,
determining the degree of elevation of a particular enzyme activity in the plasma is often useful
in evaluating the prognosis for the patient.
B. Plasma enzymes as diagnostic tools
Some enzymes show relatively high activity in only one or a few tissues.The presence of
increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue. For
example, the enzyme alanine aminotransferase (ALT, see p. 251) is abundant in the liver. The
appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. [Note:
Measurement of ALT is part of the liver function test panel.] Increases in plasma levels of
enzymes with a wide tissue distribution provide a less specific indication of the site of cellular
injury and limits their diagnostic value.
1) Aspartate amino Transferase (AST)
It is also called as Serum Glutamate Oxaloacetate Transaminase (SGOT). The level is
significantly elevated in Acute Myocardial Infarction (AMI).
Normal Value- 0-41 IU/L at 37C
In acute MI- Serum activity rises sharply within thefirst 12 hours, with a peak level at 24 hours
or over and returns to normalwithin 3-5 days. The rise depends on the extent of infarction. Reinfarctionresults in secondary rise of SGOT.
Prognostic significance- Levels> 350 IU/L are due to massive Infarction(Fatal),

> 150 IU/L are associated with high mortality and levels < 50IU/L are associated with low
mortality.
Other diseases- The rise in activity is also observed in muscle and hepatic diseases. These can
be well differentiated from simultaneous estimations of other enzyme activities like SGPT etc,
which do not show and rise in activity in Acute MI.
2) Alanine amino transferase (ALT)
It is also called serum Glutamate pyruvate transaminase (SGPT).
Normal serum level ranges between 0-45 IU/L at37oC.
Very high values are seen in Acute hepatitis, toxic or viral in origin. Both ALT and AST rise but
ALT> AST. Moderate increase may be seen in chronic liver diseases such as Cirrhosis and
Malignancy in liver. A sudden fall in AST level in hepatitis signifies bad prognosis.

Iso enzymes and their clinical significance

Isoenzymes are enzymes that catalyze identical chemical reactions but are composed of different
amino acid sequences. They are sometimes referred to as isozymes. Isoenzymes are produced by
different genes and are not redundant despite their similar functions. They occur in many tissues
throughout the body and are important for different developmental and metabolic processes.
Isoenzymes are useful biochemical markers and can be measured in the bloodstream to diagnose
medical conditions. Isoenzymes
can be differentiated from one another using gel
electrophoresis. In gel electrophoresis, isoenzyme fragments are drawn through a thick gel by an
electric charge. Each isoenzyme has a distinct charge of its own because of its unique amino acid
sequence. This enables gel electrophoresis to separate the fragments into bands for identification.
Some clinically important isoenzymes are as follows1) Creatine Kinase(CK, CPK) is an enzyme found primarily in the heart and skeletal muscles,
andto a lesser extent in the brain but not found at all in liver and kidney. Small amounts are also
found in lungs, thyroid and adrenal glands .Significant injury to any of these structures will lead
to a measurable increase in CK levels. It is not found in red blood cells and its level is not
affected by hemolysis.
NormalValue- serum activity varies from 10-50 IU/L at 30C.
Elevations are found in:

Myocardial infarction

Crushing muscular trauma

Any cardiac or muscle disease, but not myasthenia gravis or multiple sclerosis

Brain injury

Hypothyroidism

Hypokalemia

After myocardial infarction- serum value is found to increase within3-6 hours, reaches a peak
level in 24- 30 hours and returns to normal level in2-4 days (usually in 72 hours). CK is a
sensitive indicator in the early stages of myocardial ischemia. No increase in activity is found in
heart failure and coronary insufficiency. In acute MI, CPK usually rises faster than SGOT and
returns to normal faster than the SGOT.
CK/CPK
Isoenzymes
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels of CK
or CPK to determine the source of the elevation. Each isoenzyme is a dimer composed of two
protomersM (for muscles) and B( for Brain). These isoenzymes can be separated by,
Electrophoresis or by Ion exchange Chromatography. The three possible iso enzymes are;
Isoenzyme
MM(CK3)

Electrophoretic mobility
Least

MB(CK2)
BB(CK1)

Intermediate
Maximum

Tissue of origin
Mean percentage in blood
Skeletal muscle 97-100%
Heart muscle
Heart muscle
0-3%
Brain
0%

2) Lactate dehydrogenase (LDH)

Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate. LDH is
essential for anaerobic respiration. When oxygen levels are low, LDH converts pyruvate to
lactate, providing a source of muscular energy.
Normal level- 55-140IU/L at 30C. The levels in the upper range are generally seen in children.
LDH level is 100 times more inside the RBCs than in plasma, and therefore minor amount of
hemolysis results in false positive result.
In Acute MI-The serum activity rises within 12 to 24hours, attains a peak at 48 hours (2 to 4
days) reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th day. The
magnitude of rise is proportional to the extent of myocardial infarction. Serum LDH elevation
may persist for more than a week after CPK and SGOT levels have returned to normal levels.
Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemea,
hepatocellular damage, muscular dystrophies, carcinoma, leukemias, and any condition which
causes necrosis of the body cells. Since the total LDH is increased in many diseases, so the study
of Isoenzymes of LDH is of more significance.
Iso enzymes of LDH
LDH enzyme is tetramer with 4 subunits. The subunit may be either H(Heart) or M(Muscle)
polypeptide chains. These two chains are the product of 2 different genes. Although both of them
have the same molecular weight, there are minor amino acid variations. There can be 5 possible
combinations; H4, H3M1, H2M2, H1M3. M4, these are 5different types of isoenzymes seen in
all individuals.
No.
of Subunit
Electrophoretic Activity
Tissue
Percentage in human
Isoenzyme make up mobility
at at 60for origin
serum(Mean)
of
pH8.6
30

LDH-1

isoenzyme
H4

Fastest

LDH-2

H3M1

Faster

LDH-3

H2M2

Fast

LDH-4
LDH-5

H1M3
M4

Slow
Slowest

minutes
Not
destroyed
Not
destroyed.
Partly
destroyed
Destroyed
Destroyed

Heart
muscle
RBC

30%

Brain

20%

35%

Liver
10%
Skeletal 5%
Muscles

Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed in
myocardial infarction, this is called flipped pattern. These isoenzymes are separated by
cellulose acetate electrophoresis at pH 8.6.
5) Alkalinephosphatase (ALP )-is an enzyme that removes phosphate groups from organic or
inorganic compounds in the body. It is present in a number of tissues including liver, bone,
intestine, and placenta. The activity of ALP found in serum is a composite of isoenzymes from
those sites and, in some circumstances, placental or Regan isoenzymes. The optimum pH for
enzyme action varies between 9-10. It is a zinc containing metalloenzyme and is localized in the
cell membranes (ectoenzyme). It is associated with transport mechanism in the liver, kidney and
intestinal mucosa.
Normal serum Level- of ALP ranges between 40-125 IU/L. In children the upper level of
normal value may be more, because of increased osteoblastic activity.
Total Alkaline Phosphatase (ALP)
Serum ALP is of interest in the diagnosis of 2 main groups of conditions-hepatobiliary diseases
and bone diseases associated with increased osteoblastic activity.
Mild increase is observed in pregnancy, due to production of placental enzyme.
A moderate rise in ALP activity occurs in hepatic diseases such as infective hepatitis, alcoholic
hepatitis or hepatocellular carcinoma. Moderate elevation of ALP may also be seen in
otherdisorders such as Hodgkins disease, congestive heart failure, ulcerative colitis, regional
enteritis, and intra-abdominal bacterial infections.
ALP elevations tend to be more marked (more than 3-fold) in extra hepatic biliary
obstructions (eg, by stone or cancer of the head of the pancreas) than in intra hepatic
obstructions, and the morecomplete the obstruction, the greater the elevation. With obstruction,
serumALP activities may reach 10 to 12 times the upper limit of normal, returning to normal
upon surgical removal of the obstruction. The ALP response to cholestatic liver disease is similar
to the response of gamma-glutamyltransferase (GGT), but more blunted. If both GGT and ALP
are
elevated,
a
liver
source
of
the
ALP
is
likely.

The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The
main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
ALP also is elevated in disorders of the skeletal system that involve osteoblast hyperactivity and
bone remodeling, such as Pagets disease rickets and osteomalacia, fractures, and malignant
tumors.
Among bone diseases, the highest level of ALP activity is encountered in Pagets disease, as a
result of the action of the osteoblastic cells as they try to rebuild bone that is being resorbed by
the uncontrolled activity of osteoclasts. Values from 10 to 25 times the upper limit of normal are
not unusual.
Only moderate rises are observed in osteomalacia, while levels are generally normal in
osteoporosis. In rickets,levels 2 to 4 times normal may be observed. Primary and secondary
hyperparathyroidism are associated with slight to moderate elevations of ALP; the existence and
degree of elevation reflects the presence and extent of skeletal involvement.
Very high enzyme levels are present in patients with osteogenic bone cancer.
A considerable rise in ALP is seen in children following accelerated bone growth.
Patients over age 60 can have a mildly elevated alkaline phosphatase (11 times normal), while
individuals with blood types O and B can have an elevation of the serum alkaline phosphatase
aftereating a fatty meal due to the influx of intestinal alkaline phosphatase into the blood.
ALP Isoenzymes
Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis. By
starch gel electrophoresis at pH 8.6 , at least 6 isoenzyme bands can be visualized.
1) Hepatic ALP isoenzyme- moves fastest towards the anode and occupies the same position as
Alpha 2 globulins. It is associated with biliary epithelium and is elevated in cholestatic
processes. Various liver diseases (primary or secondary cancer, biliary obstruction) increase the
liver isoenzyme.
2) Bone isoenzyme- It closely follows thehepatic enzyme and occupies the beta region.
Osteoblastic bone tumors and hyperactivity of osteoblasts involved in bone remodeling (eg,
Pagets disease) increase the bone isoenzyme. Pagets disease leads to a striking, solitary
elevation of bone ALP.
3) Placental isoenzyme follows bone isoenzyme. It is heat stable isoenzyme and increases
during last six weeks of pregnancy.
4) The intestinal isoenzyme-is the slowly moving band and follows the placental isoenzyme. It
may be increased inpatients with cirrhosis and in individuals who are blood group O or B
secretors. Increased levels are also seen in patients undergoing hemodialysis.
Atypical ALP isoenzymes (Oncogenic markers)-In addition to 4 major isoenzymes, 2 more
abnormal fractions are seen associated with tumors. These are Regan and Nagao isoenzymes.

They are also called Carcino placental ALP, asthey resemble placental isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries. Highest
positivity is observed in carcinoma of ovary and uterus.
Rise in Nagao isoenzyme is observed in metastatic carcinoma of pleural surfaces and
adenocarcinoma of pancreas and bile duct.

LECTURE ONE: Enzymes: Definition and Functions

LECTURE TWO: Classification and Nomenclature Enzymes
LECTURE THREE: Mechanisms of Enzyme catalysed reactions
LECTURE FOUR: Effects of Temperature, pH, Ions,
Inhibitors and Activators On Enzymes
LECTURE FIVE: Effects of Temperature, pH, Ions, Inhibitors
and Activators On Enzymes (contd.)
LECTURE SIX: Active sites of Enzymes
LECTURE SEVEN: Vitamins and Coenzymes
LECTURE EIGHT: Enzyme Assays
LECTURE NINE: Enzyme Kinetics
LECTURE TEN: Estimation of Kinetic Parameters
LECTURE ELEVEN: Reversible and Irreversible Inhibition
LECTURE TWELVE: Production, Isolation, Purification
and Characterisation of Enzymes
LECTURE THIRTEEN: Production, Isolation, Purification and
Characterisation of Enzymes (contd.)

LECTURE ONE
ENZYMES: DEFINITION AND FUNCTIONS
WHAT ARE ENZYMES?
Enzymes are the substances that allow life to exist at earth temperatures. Without such
substances most chemical reactions that maintain a viable organism would not occur below 90oC
or 200oF. These remarkable highly specialized molecules are the key to understanding how cells
function. Most enzymes are proteins but a few are RNAs (called Ribozymes). Enzymes catalyze
(i.e., increase the rates of) chemical reactions. In enzymatic reactions, the molecules at the
beginning of the process, called substrates, are converted into different molecules, called
products. Almost all chemical reactions in a biological cell need enzymes in order to occur at
rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few
reactions from among many possibilities, the set of enzymes made in a cell determines which
metabolic pathways occur in that cell.
Like all catalysts, enzymes work by lowering the activation energy (Ea) for a reaction, thus
dramatically increasing the rate of the reaction. As a result, products are formed faster and
reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of
times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are
not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions.
However, enzymes do differ from most other catalysts in that they are highly specific for their
substrates. Enzymes are known to catalyze about 4,000 biochemical reactions.

FUNCTIONS OF ENZYMES
Enzymes serve a wide variety of functions inside living organisms. They are indispensable for
signal transduction and cell regulation, often via kinases and phosphatases. They also generate
movement, with myosin hydrolyzing ATP to generate muscle contraction and also moving cargo
around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps
involved in active transport. Enzymes are also involved in more exotic functions, such as
luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such
as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza
virus neuraminidase.
An important function of enzymes is in the digestive systems of animals. Enzymes such as
amylases and proteases break down large molecules (starch or proteins, respectively) into smaller
ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be
absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such
as maltose and eventually glucose, which can then be absorbed. Different enzymes digest
different food substances. In ruminants which have herbivorous diets, microorganisms in the gut
produce another enzyme, cellulase to break down the cellulose cell
walls of plant fiber.
Several enzymes can work together in a specific order, creating metabolic pathways. In a
metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the
catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one
enzyme can catalyze the same reaction in parallel, this can allow more complex regulation: with

for example a low constant activity being provided by one enzyme but an inducible high activity
from a second enzyme.
Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would
neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed,
a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for
example, can react directly with ATP to become phosphorylated at one or more of its carbons. In
the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is
added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs
so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the
only significant product. Consequently, the network of metabolic pathways within each cell
depends on the set of functional enzymes that are present.

CATALYSIS
In order for a reaction to occur, reactant molecules must contain sufficient energy to cross a
potential energy barrier, the activation energy. All molecules possess varying amounts of energy
depending, for example, on their recent collision history but, generally, only a few have
sufficient energy for reaction. The lower the potential energy barrier to reaction, the more
reactants have sufficient energy and, hence, the faster the reaction will occur. All catalysts,
including enzymes, function by forming a transition state, with the reactants, of lower free
energy than would be found in the uncatalysed reaction (Figure 1). Even quite modest reductions
in this potential energy barrier may produce large increases in the rate of reaction (e.g. the
activation energy for the uncatalysed breakdown of hydrogen peroxide to oxygen and water is 76
kJ M-1 whereas, in the presence of the enzyme catalase, this is reduced to 30 kJ M -1 and the rate
of reaction is increased by a factor of 10 8, sufficient to convert a reaction time measured in years
into one measured in seconds).

Figure 1. A schematic diagram showing the free energy profile of the course of an enzyme
catalysed reaction involving the formation of enzyme-substrate (ES) and enzyme-product (EP)
complexes, i.e.
The catalysed reaction pathway goes through the transition states TS c1, TSc2 and TSc3, with
standard free energy of activation Gc*, whereas the uncatalysed reaction goes through the
transition state TSu with standard free energy of activation G u*. In this example the rate limiting
step would be the conversion of ES into EP. Reactions involving several substrates and products,
or more intermediates, are even more complicated. The schematic profile for the uncatalysed
reaction is shown as the dashed line. It should be noted that the catalytic effect only concerns the
lowering of the standard free energy of activation from G u* to Gc* and has no effect on the
overall free energy change (i.e.. the difference between the initial and final states) or the related
equilibrium constant.

REFERENCES
1. Smith, A. L. (Ed) (1997). Oxford dictionary of biochemistry and molecular biology. Oxford
[Oxfordshire]: Oxford University Press.
2. Lilley, D. (2005). "Structure, folding and mechanisms of ribozymes". Curr Opin Struct Biol 15
(3): 31323.
3. Cech, T. (2000). "Structural biology. The ribosome is a ribozyme". Science 289 (5481): 8789.

4. Garcia-Viloca M., Gao J., Karplus M., Truhlar D. G. (2004). "How enzymes work: analysis
by modern rate theory and computer simulations". Science 303 (5655): 18695.

QUESTIONS
1. Define an Enzyme
2. State at least 5 functions of Enzymes.
3. How does an enzyme carry out catalysis?

LECTURE TWO
CLASSIFICATION AND NOMENCLATURE ENZYMES
INTRODUCTION
Enzyme classification and nomenclature is a system that allows the unambiguous identification

of enzymes in terms of the reactions they catalyse. This relies on a numerical system to class
enzymes in groups according to the types of reaction catalysed and systematic naming that
describes the chemical reaction involved.
Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin, most
enzyme names end in "ase". The International Union of Biochemistry (I.U.B.) initiated standards
of enzyme nomenclature which recommend that enzyme names indicate both the substrate acted
upon and the type of reaction catalyzed. Under this system, the enzyme uricase is called urate: O2
oxidoreductase, while the enzyme glutamic oxaloacetic transaminase (GOT) is called Laspartate: 2-oxoglutarate aminotransferase.
The International Union of Biochemistry and Molecular Biology have developed a nomenclature
for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers
preceded by "EC". The first number broadly classifies the enzyme based on its mechanism.
According to the naming conventions, enzymes are generally classified into six main family
classes and many sub-family classes.
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
EC 5 Isomerases: catalyze isomerization changes within a single molecule
EC 6 Ligases: join two molecules with covalent bonds.
The IUBMB committee also defines subclasses and sub-subclasses. Each enzyme is assigned an
EC (Enzyme Commission) number. For example, the EC number of catalase is EC1.11.1.6. The
first digit indicates that the enzyme belongs to oxidoreductase (class 1). Subsequent digits
represent subclasses and sub-subclasses.

SCHEME FOR THE CLASSIFICATION OF ENZYMES AND THE

GENERATION OF EC NUMBERS
The first Enzyme Commission, in its report in 1961, devised a system for classification of
enzymes that also serves as a basis for assigning code numbers to them. These code numbers,
prefixed by EC, which are now widely in use, contain four elements separated by points, with the
following meaning:
(i) the first number shows to which of the six main divisions (classes) the enzyme belongs,
(ii) the second figure indicates the subclass,
(iii) the third figure gives the sub-subclass,
(iv) the fourth figure is the serial number of the enzyme in its sub-subclass.
The subclasses and sub-subclasses are formed according to principles indicated below.
The main divisions and subclasses are:

Class 1. Oxidoreductases.
To this class belong all enzymes catalysing oxidoreduction reactions. The substrate that is
oxidized is regarded as hydrogen donor. The systematic name is based on donor:acceptor
oxidoreductase. The common name will be dehydrogenase, wherever this is possible; as an
alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor.
The second figure in the code number of the oxidoreductases, unless it is 11, 13, 14 or 15,
indicates the group in the hydrogen (or electron) donor that undergoes oxidation: 1 denotes a
-CHOH- group, 2 a -CHO or -CO-COOH group or carbon monoxide, and so on, as listed in the
key.

The third figure, except in subclasses EC 1.11, EC 1.13, EC 1.14 and EC 1.15, indicates the type
of acceptor involved: 1 denotes NAD(P)+, 2 a cytochrome, 3 molecular oxygen, 4 a disulfide, 5 a
quinone or similar compound, 6 a nitrogenous group, 7 an iron-sulfur protein and 8 a flavin. In
subclasses EC 1.13 and EC 1.14 a different classification scheme is used and sub-subclasses are
numbered from 11 onwards.
It should be noted that in reactions with a nicotinamide coenzyme this is always regarded as
acceptor, even if this direction of the reaction is not readily demonstrated. The only exception is
the subclass EC 1.6, in which NAD(P)H is the donor; some other redox catalyst is the acceptor.
Although not used as a criterion for classification, the two hydrogen atoms at carbon-4 of the
dihydropyridine ring of nicotinamide nucleotides are not equivalent in that the hydrogen is
transferred stereospecifically.

Class 2. Transferases.
Transferases are enzymes transferring a group, e.g. a methyl group or a glycosyl group, from one
compound (generally regarded as donor) to another compound (generally regarded as acceptor).
The systematic names are formed according to the scheme donor:acceptor grouptransferase. The
common names are normally formed according to acceptor grouptransferase or donor
grouptransferase. In many cases, the donor is a cofactor (coenzyme) charged with the group to
be transferred. A special case is that of the transaminases (see below).
Some transferase reactions can be viewed in different ways. For example, the enzyme-catalysed
reaction
X-Y + Z = X + Z-Y
may be regarded either as a transfer of the group Y from X to Z, or as a breaking of the X-Y
bond by the introduction of Z. Where Z represents phosphate or arsenate, the process is often
spoken of as 'phosphorolysis' or 'arsenolysis', respectively, and a number of enzyme names based
on the pattern of phosphorylase have come into use. These names are not suitable for a
systematic nomenclature, because there is no reason to single out these particular enzymes from
the other transferases, and it is better to regard them simply as Y-transferases.
In the above reaction, the group transferred is usually exchanged, at least formally, for hydrogen,
so that the equation could more strictly be written as:
X-Y + Z-H = X-H + Z-Y.
Another problem is posed in enzyme-catalysed transaminations, where the -NH 2 group and -H
are transferred to a compound containing a carbonyl group in exchange for the =O of that group,
according to the general equation:
R1-CH(-NH2)-R2 + R3-CO-R4 R1-CO-R2 + R3-CH(-NH2)-R4.
The reaction can be considered formally as oxidative deamination of the donor (e.g. amino acid)
linked with reductive amination of the acceptor (e.g. oxo acid), and the transaminating enzymes
(pyridoxal-phosphate proteins) might be classified as oxidoreductases. However, the unique
distinctive feature of the reaction is the transfer of the amino group (by a well-established
mechanism involving covalent substrate-coenzyme intermediates), which justified allocation of
these enzymes among the transferases as a special subclass (EC 2.6.1, transaminases).
The second figure in the code number of transferases indicates the group transferred; a onecarbon group in EC 2.1, an aldehydic or ketonic group in EC 2.2, an acyl group in EC 2.3 and so
on.
The third figure gives further information on the group transferred; e.g. subclass EC 2.1 is
subdivided into methyltransferases (EC 2.1.1), hydroxymethyl- and formyltransferases (EC

2.1.2) and so on; only in subclass EC 2.7, does the third figure indicate the nature of the acceptor
group.

Class 3. Hydrolases.
These enzymes catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds,
including phosphoric anhydride bonds. Although the systematic name always includes hydrolase,
the common name is, in many cases, formed by the name of the substrate with the suffix -ase. It
is understood that the name of the substrate with this suffix means a hydrolytic enzyme.
A number of hydrolases acting on ester, glycosyl, peptide, amide or other bonds are known to
catalyse not only hydrolytic removal of a particular group from their substrates, but likewise the
transfer of this group to suitable acceptor molecules. In principle, all hydrolytic enzymes might
be classified as transferases, since hydrolysis itself can be regarded as transfer of a specific group
to water as the acceptor. Yet, in most cases, the reaction with water as the acceptor was
discovered earlier and is considered as the main physiological function of the enzyme. This is
why such enzymes are classified as hydrolases rather than as transferases.
Some hydrolases (especially some of the esterases and glycosidases) pose problems because they
have a very wide specificity and it is not easy to decide if two preparations described by different
authors (perhaps from different sources) have the same catalytic properties, or if they should be
listed under separate entries. An example is vitamin A esterase (formerly EC 3.1.1.12, now
believed to be identical with EC 3.1.1.1). To some extent the choice must be arbitrary; however,
separate entries should be given only when the specificities are sufficiently different.
Another problem is that proteinases have 'esterolytic' action; they usually hydrolyse ester bonds
in appropriate substrates even more rapidly than natural peptide bonds. In this case, classification
among the peptide hydrolases is based on historical priority and presumed physiological
function.
The second figure in the code number of the hydrolases indicates the nature of the bond
hydrolysed; EC 3.1 are the esterases; EC 3.2 the glycosylases, and so on.
The third figure normally specifies the nature of the substrate, e.g. in the esterases the carboxylic
ester hydrolases (EC 3.1.1), thiolester hydrolases (EC 3.1.2), phosphoric monoester hydrolases
(EC 3.1.3); in the glycosylases the O-glycosidases (EC 3.2.1), N-glycosylases (EC 3.2.2), etc.
Exceptionally, in the case of the peptidyl-peptide hydrolases the third figure is based on the
catalytic mechanism as shown by active centre studies or the effect of pH.

Class 4. Lyases.
Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by elimination, leaving double
bonds or rings, or conversely adding groups to double bonds. The systematic name is formed
according to the pattern substrate group-lyase. The hyphen is an important part of the name, and
to avoid confusion should not be omitted, e.g. hydro-lyase not 'hydrolyase'. In the common
names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2,
aldehyde, or water) are used. In cases where the reverse reaction is much more important, or the
only one demonstrated, synthase (not synthetase) may be used in the name. Various subclasses of
the lyases include pyridoxal-phosphate enzymes that catalyse the elimination of a - or substituent from an -amino acid followed by a replacement of this substituent by some other
group. In the overall replacement reaction, no unsaturated end-product is formed; therefore, these
enzymes might formally be classified as alkyl-transferases (EC 2.5.1...). However, there is ample
evidence that the replacement is a two-step reaction involving the transient formation of enzyme-

bound ,(or ,)-unsaturated amino acids. According to the rule that the first reaction is
indicative for classification, these enzymes are correctly classified as lyases. Examples are
tryptophan synthase (EC 4.2.1.20) and cystathionine -synthase (EC 4.2.1.22).
The second figure in the code number indicates the bond broken: EC 4.1 are carbon-carbon
lyases, EC 4.2 carbon-oxygen lyases and so on.
The third figure gives further information on the group eliminated (e.g. CO2 in EC 4.1.1, H2O in
EC 4.2.1).

Class 5. Isomerases.
These enzymes catalyse geometric or structural changes within one molecule. According to the
type of isomerism, they may be called racemases, epimerases, cis-trans-isomerases, isomerases,
tautomerases, mutases or cycloisomerases.
In some cases, the interconversion in the substrate is brought about by an intramolecular
oxidoreduction (EC 5.3); since hydrogen donor and acceptor are the same molecule, and no
oxidized product appears, they are not classified as oxidoreductases, even though they may
contain firmly bound NAD(P)+.
The subclasses are formed according to the type of isomerism, the sub-subclasses to the type of
substrates.

Class 6. Ligases.
Ligases are enzymes catalysing the joining together of two molecules coupled with the
hydrolysis of a diphosphate bond in ATP or a similar triphosphate. The systematic names are
formed on the system X:Y ligase (ADP-forming). In earlier editions of the list the term
synthetase has been used for the common names. Many authors have been confused by the use of
the terms synthetase (used only for Group 6) and synthase (used throughout the list when it is
desired to emphasis the synthetic nature of the reaction). Consequently NC-IUB decided in 1983
to abandon the use of synthetase for common names, and to replace them with names of the type
X-Y ligase. In a few cases in Group 6, where the reaction is more complex or there is a common
name for the product, a synthase name is used (e.g. EC 6.3.2.11 and EC 6.3.5.1).

REFERENCES
1. Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1979).
"Units of Enzyme Activity". Eur. J. Biochem. 97 (2): 31920.
2. Bennett, T. P., and Frieden, E.: (1969). Modern Topics in Biochemistry, pg. 43-45, Macmillan,
London.

QUESTIONS
1. Justify the necessity for the classification and nomenclature of enzymes.
2. Explain the modalities for classifying and naming enzymes.

LECTURE THREE
MECHANISMS OF ENZYME CATALYSED REACTIONS
MECHANISM OF SUBSTRATE BINDING
THE LOCK AND KEY MODEL
The high specificity and efficiency of enzymes can be explained by the manner that they
associate with the reactant molecules called the substrate. One of the first theories or models that

help to explain this phenomenally efficient catalytic efficiency of enzymes is called the "lock and
key" model. According to this model, each enzyme molecule may have as few as one active site
on the surface of the enzyme molecule itself. An active site is an indentation or cavity whereby a
reactant molecule (substrate) is attracted to. This binding results in enzyme-substrate complex.
The polar and non-polar groups of the active site attract compatible groups on the substrate
molecule so that the substrate molecule can effectively lock into the cavity and position itself for
the necessary collisions and bond breaks and formations that must take place for successful
conversion to a product molecule. Once the product molecule has been formed the electrical
attractions that made the substrate molecule adhere to the active site no longer are present, and
the product molecule can disengage itself from the active site thus freeing the site for another
incoming substrate molecule. This process occurs in a highly efficient manner hundreds or even
thousands of times in a short time span.

LIMITATION OF THE LOCK AND KEY MODEL

The Lock and Key model assumes that molecules that lock into the active site must form a
perfect fit. Also the assumption is that the active site conformation is rigid. Evidence does not
support these assumptions. For example, certain "bogus" molecules can lock into the active site
even though the bogus molecules have a different shape compared to the true substrate molecule.
This has the effect of inhibiting enzyme activity. This does not seem to support the rigid active
site assumption in the lock and key model. Furthermore, small temperature changes and small
changes in pH will not result in the enzyme being inhibited from catalyzing its intended reaction.
The occurance of pH and temperature ranges of optimum enzyme activity does not support the
assumptions made by the lock and key model of rigid active site cavities. Modification of the
lock and key model is necessary to account for the occurance of pH and temperature ranges of
optimum enzyme activity and to explain why other molecules can effectively block the active
site.

THE INDUCED FIT MODEL

The favored model for the enzyme-substrate interaction is the induced fit model. This model
proposes that the initial interaction between enzyme and substrate is relatively weak, but that
these weak interactions rapidly induce conformational changes in the enzyme that strengthen
binding. This is a modification of the lock and key model. It assumes that the active site has a
certain amount of elasticity whereby the active site can expand or contract in a limited way in
order to accommodate the substrate molecule. The analogy is like a hand fitting into a glove. The
glove adjusts in shape and size to fit various sized hands within a certain range. This tolerance
would explain why bogus molecules of slightly different size compared to the true substrate
molecule can still be accommodated by the elastic active site. Small changes in temperature
would distort the active site conformation but not so much that the active site could not still
accommodate the substrate molecular size. PH changes which would also change the active site
conformation but not so much that the active site could not flexibly accommodate the substrate
molecule. The Induced Fit Model seems to explain why there is some flexibility in the ability of
the active site to accommodate other molecules and at limited temperature and pH ranges.

]
Figure 2. Diagrams to show the induced fit hypothesis of enzyme action.

MECHANISMS OF TRANSITION STATE STABILIZATION

INTRODUCTION
According to the Induced fit model, the binding of substrates to the active site brings about
conformational changes in the active site. These conformational changes also bring catalytic
residues in the active site close to the chemical bonds in the substrate that will be altered in the
reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the
reaction's transition state, by providing an alternative chemical pathway for the reaction. There
are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier"
mechanism:

CATALYSIS BY BOND STRAIN

This is the principal effect of induced fit binding, where the affinity of the enzyme to the
transition state is greater than its affinity to the substrate itself. This induces structural
rearrangements which strain substrate bonds into a position closer to the conformation of the
transition state, so lowering the energy difference between the substrate and transition state and
helping catalyze the reaction.
However, the strain effect is, in fact, a ground state destabilization effect, rather than transition
state stabilization effect. Furthermore, enzymes are very flexible and they cannot apply large
strain effect.
In addition to bond strain in the substrate, bond strain may also be induced within the enzyme
itself to activate residues in the active site.
For example:
Substrate, bound substrate, and transition state conformations of lysozyme.

The substrate, on binding, is distorted from the typical 'chair' hexose ring into the 'sofa'
conformation, which is similar in shape to the transition state.

CATALYSIS BY PROXIMITY AND ORIENTATION

This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical
groups and hold them close together. This reduces the entropy of the reactants and thus makes
reactions such as ligations or addition reactions more favorable, there is a reduction in the overall
loss of entropy when two reactants become a single product.
This effect is analogous to an effective increase in concentration of the reagents. The binding of
the reagents to the enzyme gives the reaction intramolecular character, which gives a massive
rate increase.
For example:
Similar reactions will occur far faster if the reaction is intramolecular.

The effective concentration of acetate in the intramolecular reaction can be estimated as k2/k1
= 2 x 105 Molar.

CATALYSIS INVOLVING
(ACID/BASE CATALYSIS)

PROTON

DONORS

OR

ACCEPTORS

Proton donors and acceptors, i.e. acids and bases, may donate and accept protons in order to
stabilize developing charges in the transition state. This typically has the effect of activating

nucleophile and electrophile groups, or stabilizing leaving groups. Histidine is often the residue
involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore
both accept and donate protons.
Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa.
This alteration of pKa is possible through the local environment of the residue.
Conditions
Acids
Bases
Hydrophobic environment

Increase
pKa

Decrease
pKa

Adjacent residues of like

charge

Increase
pKa

Decrease
pKa

Salt bridge (and hydrogen

bond) formation

Decrease
pKa

Increase
pKa

The pKa can be modified significantly by the environment, to the extent that residues which are
basic in solution may act as proton donors, and vice versa.
For example:
Serine protease catalytic mechanism

The initial step of the serine protease catalytic mechanism involves the histidine of the active
site accepting a proton from the serine residue. This prepares the serine as a nucleophile to
attack the amide bond of the substrate. This mechanism includes donation of a proton from
serine (a base, pKa 14) to histidine (an acid, pKa 6), made possible due to the local
environment of the bases.
It is important to clarify that the modification of the pKas is a pure part of the electrostatic
mechanism. Furthermore, the catalytic effect of the above example is mainly associated with the
reduction of the pKa of the oxy anion and the increase in the pKa of the histidine, while the
proton transfer from the serine to the histidine is not catalyzed significantly, since it is not the

rate determining barrier.

ELECTROSTATIC CATALYSIS
Stabilization of charged transition states can also be by residues in the active site forming ionic
bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come
from acidic or basic side chains found on amino acids such as lysine, arginine, aspartic acid or
glutamic acid or come from metal cofactors such as zinc. Metal ions are particularly effective
and can reduce the pKa of water enough to make it an effective nucleophile.
Systematic computer simulation studies established that electrostatic effects give, by far, the
largest contribution to catalysis. In particular, it has been found that enzyme provides an
environment which is more polar than water, and that the ionic transition states are stabilized by
fixed dipoles. This is very different from transition state stabilization in water, where the water
molecules must pay with "reorganization energy" in order to stabilize ionic and charged states.
Thus, the catalysis is associated with the fact that the enzyme polar groups are preorganized.

For example:
Carboxypeptidase catalytic mechanism

The tetrahedral intermediate is stabilised by a partial ionic bond between the Zn2+ ion and the
negative charge on the oxygen.

COVALENT CATALYSIS
Covalent catalysis involves the substrate forming a transient covalent bond with residues in the
active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and
helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a
later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in
enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is

formed. Schiff base formation using the free amine from a lysine residue is another mechanism,
as seen in the enzyme aldolase during glycolysis.
Some enzymes utilize non-amino acid cofactors such as pyridoxal phosphate (PLP) or thiamine
pyrophosphate (TPP) to form covalent intermediates with reactant molecules. Such covalent
intermediates function to reduce the energy of later transition states, similar to how covalent
intermediates formed with active site amino acid residues allow stabilization, but the capabilities
of cofactors allow enzymes to carryout reactions that amino acid side residues alone could not.
Enzymes utilizing such cofactors include the PLP-dependent enzyme aspartate transaminase and
the TPP-dependent enzyme pyruvate dehydrogenase.
It is important to clarify that covalent catalysis does correspond in most cases to simply the use
of a specific mechanism rather than to true catalysis.

QUANTUM TUNNELING
These traditional "over the barrier" mechanisms have been challenged in some cases by models
and observations of "through the barrier" mechanisms (quantum tunneling). Some enzymes
operate with kinetics which are faster than what would be predicted by the classical G . In
"through the barrier" models, a proton or an electron can tunnel through activation barriers.
Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine
dehydrogenase.
Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since
the tunneling contributions are similar in the catalyzed and the uncatalyzed reactions in solution.
However, the tunneling contribution (typically enhancing rate constants by a factor of ~1000
compared to the rate of reaction for the classical 'over the barrier' route) is likely crucial to the
viability of biological organisms. This emphasizes the general importance of tunneling reactions
in biology.
REFERENCES
1. Copeland, Robert A. Enzymes. Wiley-VCH, Inc., 2000. 154-70.
2. Fersht, Alan. Enzyme Structure and Mechanism. W.H. Freeman and Company, 1985. 47-77.
Milton H. Saier, Jr. Enzymes in Metabolic Pathways,

QUESTIONS
1. Explain the mechanisms of enzyme binding.
2. Why is the Induced-fit model preferable to the Lock and Key model?
3. Explain the mechanisms of transition state stabilization.

LECTURE FOUR
ENZYME KINETICS
STEADY STATE KINETICS
In most cases, an enzyme converts one chemical (the substrate), into another (the product). A
graph of product concentration vs. time follows three phases as shown in the following graph.

THE TRANSCIENT STATE (PHASE 1)

At very early time points, the rate of product accumulation increases over time. Special
techniques are needed to study the early kinetics of enzyme action; since this transient phase
usually lasts less than a second (the figure greatly exaggerates the first phase).

THE STEADY STATE (PHASE 2)

For an extended period of time, the product concentration increases linearly with time.
At later times, the substrate is depleted, so the curve starts to level off. Eventually the
concentration of product reaches a plateau and doesn't change with time.
It is difficult to fit a curve to a graph of product as a function of time, even if you use a simplified
model that ignores the transient phase and assumes that the reaction is irreversible. The model
simply cannot be solved to an equation that expresses product concentration as a function of
time. To fit these kind of data (called an enzyme progress curve) you need to use a program that
can fit data to a model defined by differential equations or by an implicit equation. Prism cannot
do this.
Rather than fit the enzyme progress curve, most analyses of enzyme kinetics fit the initial
velocity of the enzyme reaction as a function of substrate concentration. The velocity of the
enzyme reaction is the slope of the linear phase, expressed as amount of product formed per
time. If the initial transient phase is very short, you can simply measure product formed at a
single time, and define the velocity to be the concentration divided by the time interval.
This lecture considers data collected only in the second phase. The terminology describing these
phases can be confusing. The second phase is often called the "initial rate", ignoring the short
transient phase that precedes it. It is also called "steady state", because the concentration of
enzyme-substrate complex doesn't change. However, the concentration of product accumulates,
so the system is not truly at steady state until, much later, the concentration of product truly
doesn't change over time.
Enzyme velocity as a function of substrate concentration
If you measure enzyme velocity at many different concentrations of substrate, the graph
generally looks like this:

Enzyme velocity as a function of substrate concentration often follows the Michaelis-Menten

equation:

Vmax is the limiting velocity as substrate concentrations get very large. Vmax (and V) are
expressed in units of product formed per time. If you know the molar concentration of enzyme,
you can divide the observed velocity by the concentration of enzyme sites in the assay, and
express Vmax as units of moles of product formed per second per mole of enzyme sites. This is
the turnover number, the number of molecules of substrate converted to product by one enzyme
site per second. In defining enzyme concentration, distinguish the concentration of enzyme
molecules and concentration of enzyme sites (if the enzyme is a dimer with two active sites, the
molar concentration of sites is twice the molar concentration of enzyme).
KM is expressed in units of concentration, usually in Molar units. KM is the concentration of
substrate that leads to half-maximal velocity. To prove this, set [S] equal to KM in the equation
above. Cancel terms and you'll see that V=Vmax/2.
The meaning of KM
To understand the meaning of Km, you need to have a model of enzyme action. The simplest
model is the classic model of Michaelis and Menten, which has proven useful with many kinds
of enzymes.

The substrate (S) binds reversibly to the enzyme (E) in the first reaction. In most cases, you can't
measure this step. What you measure is production of product (P), created by the second
reaction.
From the model, we want to derive an equation that describes the rate of enzyme activity
(amount of product formed per time interval) as a function of substrate concentration.
The rate of product formation equals the rate at which ES turns into E+P, which equals k2 times
[ES]. This equation isn't helpful, because we don't know ES. We need to solve for ES in terms of
the other quantities. This calculation can be greatly simplified by making two reasonable

assumptions. First, we assume that the concentration of ES is steady during the time intervals
used for enzyme kinetic work. That means that the rate of ES formation, equals the rate of ES
dissociation (either back to E+S or forward to E+P). Second, we assume that the reverse reaction
(formation of ES from E+P) is negligible, because we are working at early time points where the
concentration of product is very low.

We also know that the total concentration of enzyme, Etotal, equals ES plus E. So the equation
can be rewritten.
Solving for ES:

The velocity of the enzyme reaction therefore is:

Finally, define Vmax (the velocity at maximal concentrations of substrate) as k2 times Etotal,
and KM, the Michaelis-Menten constant, as (k2+k-1)/k1. Substituting:

Note that Km is not a binding constant that measures the strength of binding between the enzyme
and substrate. Its value includes the affinity of substrate for enzyme, but also the rate at which
the substrate bound to the enzyme is converted to product. Only if k2 is much smaller than k-1
will KM equal a binding affinity.The Michaelis-Menten model is too simple for many purposes.
The Briggs-Haldane model has proven more useful:

Under the Briggs-Haldane model, the graph of enzyme velocity vs. substrate looks the same as
under the Michaelis-Menten model, but KM is defined as a combination of all five of the rate
constants in the model.
Assumptions of enzyme kinetic analyses
Standard analyses of enzyme kinetics (the only kind discussed here) assume:
The production of product is linear with time during the time interval
used.
The concentration of substrate vastly exceeds the concentration of enzyme. This means
that the free concentration of substrate is very close to the concentration you added, and
that substrate concentration is constant throughout the assay.

A single enzyme forms the

product.
There is negligible spontaneous creation of product without
enzyme

ESTIMATION OF KINETIC PARAMETERS

Determining KM and Vmax experimentally
To characterize an enzyme-catalyzed reaction KM and Vmax need to be determined. The way
this is done experimentally is to measure the rate of catalysis (reaction velocity) for different
substrate concentrations. In other words, determine V at different values of [S]. Then plotting
1/V vs. 1/[S] we should obtain a straight line described by Lineweaver-Burk

LECTURE FIVE

MICHAELIS- MENTEN KINETICS

The primary function of enzymes is to enhance rates of reactions so that they are compatible with
the needs of the organism. To know about functioning of enzymes, a kinetic description of their
activity is needed. The study of chemical reactions catalyzed by enzymes is known as enzyme
kinetics.
As enzyme-catalysed reactions are saturable, their rate of catalysis does not show a linear
response to increasing substrate. If the initial rate of the reaction is measured over a range of
substrate concentrations (denoted as [S]), the reaction rate (V0) increases as [S] increases, as

shown in figure-4.

Figure 4: Saturation curve for an enzyme

which obeys Michaelis-Menten kinetics

However, as [S] gets higher, the enzyme becomes saturated with substrate and the rate
reaches Vmax, the enzyme's maximum rate. In 1913, Leonor Michaelis and Maud Menten
proposed a simple model to account for these kinetic characteristics. In this model, a specific ES
complex is a necessary intermediate in catalysis. Consider an enzyme that catalyzes the S to P
by the following pathway:

An enzyme E combines with substrate S to form an ES complex, with a rate constant k1. The
ES complex can dissociate to E and S with a rate constant k-1, or it can proceed to form product
P, with a rate constant k2. It can also be assumed that almost none of the product reverts to the
initial substrate, a condition that holds in the initial stage of a reaction before the concentration of
product is appreciable.
The catalytic rate is equal to the product of the concentration of the ES complex and k2.
V0 = k2 [ES]-------------Eqn (1)
The rate of formation of ES is given by:
[ES] = k1 [E] [S]-----------Eqn (2)
The rate of breakdown of ES is given by:
[ES] = (k-1 + k2) [ES]------------Eqn (3)
Considering a steady state, [ES], stay the same even if the concentrations of starting materials
and products are changing. This occurs when the rates of formation and breakdown of the ES
complex are equal. Therefore equating equations 2 and 3,

k1 [E] [S] = (k-1 + k2) [ES], which can be modified to

( [E] [S] ) / [ES] =

( k-1 + k2) / k1--------------Eqn (4)

Equation 4 can be simplified by defining a new constant, KM, called the Michaelis constant:
KM = ( k-1 + k2) / k1----------------------Eqn (5)
KM is an important characteristic of enzyme-substrate interactions and is independent of enzyme
and substrate concentrations.
Considering equations 4 and 5, [ES] can be determined, which is
[ES] = ( [E] [S] ) / KM -------------------------Eqn (6)
The concentration of uncombined substrate [S] is very nearly equal to the total substrate
concentration, provided that the concentration of enzyme is much lower than that of substrate.
The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E]T
minus the concentration of the ES complex. Therefore
[E] = [E]T - [ES] ------------------------- eqn (7)
Equation 6 can be written as
[ES] = ( ( [E]T - [ES] ) [S] ) / KM -------------------------Eqn (8)
Solving equation 8,
[ES] =

( ( [E]T [S] ) / KM ) / (1 + [S] / KM ) -------------------Eqn (9)

Equation 9 can be modified as

[ES] = [E]T ( [S] / [S] + KM )
Substituting the value of [ES] from equation 9 in equation 1,
V0 = k2 [E]T ( [S] / [S] + KM ) -------------------Eqn (10)
The maximal rate, Vmax, is attained when the catalytic sites on the enzyme are saturated with
substrate, that means when
[ES] = [E]T.
Therefore,
Vmax = k2 [E]T --------------------------Eqn (11)
Putting the value of Vmax from equation 11 in equation 10, we get

V0 = Vmax ( [S] / [S] + KM ) ------------------- The Michaelis Menten equation.

The equation states that, at very low substrate concentration, when [S] is much less than KM,
Then,V0 = (Vmax / KM) [S]; that is, the rate is directly proportional to the substrate concentration.
At high substrate concentration, when [S] is much greater than KM, Then V0 = Vmax ; that is, the
rate is maximal, independent of substrate concentration.

LECTURE SIX
ALLOSTERIC ENZYME
Allosteric enzymes are enzymes that change their conformational ensemble upon binding of an
effector/regulator, which results in an apparent change in binding affinity at a different ligand
binding site. This "action at a distance" through binding of one ligand affecting the binding of
another at a distinctly different site, is the essence of the allosteric concept. Allostery plays a
crucial role in many fundamental biological processes, including but not limited to cell signaling
and the regulation of metabolism. Allosteric enzymes need not be oligomers as previously
thought, and in fact many systems have demonstrated allostery within single enzymes.
Allosteric enzyme can be in two states; active and inactive. In the first instance, the process is
relatively normal, where the substrate binds to its binding Site and carries out its reaction. In the
second instance, the substrate is unable to attach itself to the binding Site. This is due to a
modulator molecule which alters the shape of the binding Site, making it inactive allosteric
enzyme.

Properties and Evolution Of Allosteric Enzymes

The purpose of regulatory enzymes is to permit control of the rate at a specific metabolic step.
There are two major groups of allosteric enzymes. Both employ a change in affinity for the
substrate to alter activity. One of the groups experiences only a change in affinity for the
principal substrate, while keeping the maximum rate fairly constant (K-type enzymes). The
second group also demonstrates significant changes in affinity for the main substrate, and in
addition has large changes in the maximum rate (V-type enzymes). The greatest changes in Vmax
are for enzymes that act as regulatory switches.

Kinetic properties
Whereas enzymes without coupled domains/subunits display normal Michaelis-Menten kinetics,
most allosteric enzymes have multiple coupled domains/subunits and show cooperative binding.
Generally speaking, such cooperativity results in allosteric enzymes displaying a sigmoidal
dependence on the concentration of their substrates in positively cooperative systems. This
allows most allosteric enzymes to greatly vary catalytic output in response to small changes in
effector concentration. Effector molecules, which may be the substrate itself (homotropic
effectors) or some other small molecule (heterotropic effector), may cause the enzyme to become
more active or less active by redistributing the ensemble between the higher affinity and lower
affinity states. The binding sites for heterotropic effectors, called allosteric sites, are usually
separate from the active site yet thermodynamically coupled.
Allosteric enzymes consists of multiple subunits. Binding of a substrate (or effector) molecule at
one subunit induces a conformational change in the subunit. This causes conformational changes
in the other subunits. Thus a significant part of the binding energy of the substrate is used to
change the conformation of the protein complex. Binding of a substrate molecule at another
binding site does not need alter the structure of the subunit anymore, thus the binding affinity
increases. This property changes the curve of activity (v0) versus substrate concentration from a
hyperbole (Michaelis-Menten kinetics) into an "S" shape, sigmoidal curve. Sigmoidal behaviour
can be described with the Hill equation: v0 = V MAX*[s]n/([s]n + K0.5n)

Sigmoid Kinetics
A significant portion of enzymes function such that their properties can be studied using the

Michaelis-Menten equation. However, a particular class of enzymes exhibit kinetic properties

that cannot be studied using the Michaelis-Menten equation. The rate equation of these unique
enzymes is characterized by an S-shaped sigmoidal curve, which is different from the majority
of enzymes whose rate equation exhibits hyberbolic curves.

Introduction
Sigmoidal kinetic profiles are the result of enzymes that demonstrate positive cooperative
binding. Cooperativity refers to the observation that binding of the substrate or ligand at
one binding site affects the affinity of other sites for their substrates. For enzymatic
reactions with multiple substrate binding sites, this increased affinity for the substrate causes a
rapid and coordinated increase in the velocity of the reaction at higher \([S]\) until \(V_{max}\)
is achieved. Plotting the \(V_0\) vs. \([S]\) for a cooperative enzyme, we observe the
characteristic sigmoidal shape with low enzyme activity at low substrate concentration and a
rapid and immediate increase in enzyme activity to \(V_{max}\) as \([S]\) increases. The
phenomenon of cooperativity was initially observed in the oxygen-hemoglobin interaction that
functions in carrying oxygen in blood. Positive cooperativity implies allosteric binding binding
of the ligand at one site increases the enzymes affinity for another ligand at a site different from
the other site (Figure 2). Enzymes that demonstrate cooperativity are defined as allosteric. There
are several types of allosteric interactions: (positive & negative) homotropic and heterotropic

.
Figure 1: Rate of Reaction (velocity) vs. Substrate Concentration.
Positive and negative allosteric interactions (as illustrated through the phenomenon of
cooperativity) refer to the enzyme's binding affinity for other ligands at other sites, as a result of
ligand binding at the initial binding site. When the ligands interacting are all the same
compounds, the effect of the allosteric interaction is considered homotropic. When the ligands
interacting are different, the effect of the allosteric interaction is considered heterotropic. It is
also very important to remember that allosteric interactions tend to be driven by ATP hydrolysis.
Hill Coefficient
The degree of cooperativity is determined by Hill equation (Equation 1) for non-MichaelisMenten kinetics. The Hill equation accounts for allosteric binding at sites other than the active

site. n is the "Hill coefficient." When n < 1, there is negative cooperativity; When n = 1, there is
no cooperativity; When n > 1, there is positive cooperativity
\[ \theta = \dfrac{[L]^n}{K_d+[L]^n} = \dfrac{[L]^n}{K_a^n+[L]^n} \tag{1}\]
where
\( \theta \) is the fraction of ligand binding sites filled
\([L]\) is the ligand concentration
\(K_d\) is the apparent dissociation constant derived from the law of mass action
(equilibrium constant for dissociation)
\(K_a\) is the ligand concentration producing half occupation (ligand concentration
occupying half of the binding sides), that is also the microscopic dissociation constant
\(n\) is the Hill coefficient that describes the cooperativity
Taking the logarithm of both sides of the equation leads to an alternative formulation of the HIll
Equation.
\[ \log \left( \dfrac{\theta}{1-\theta} \right) = n\log [L] - \log K_d \tag{2}\]

Deviation of allosteric enzymes from Michaelis-Menten kinetics

The relationship between V0 and [S] in allosteric enzymes is different from that of MichaelisMenten kinetics. They do exhibit saturation with the substrate when [S] is sufficiently high, but
for some allosteric enzymes, plots of V0 versus [S] produce a sigmoid saturation curve (figure-5)
rather than the hyperbolic curve of non-regulatory enzymes (figure-4). In allosteric enzymes, the
binding of substrate to one active site can affect the properties of other active sites in the same
enzyme molecule. The binding of substrate to one active site of the enzyme facilitates substrate
binding to the other active sites, and this cooperatively results in a sigmoidal plot of V0 versus
[S]. On the sigmoid saturation curve, the value of [S], at half maximal V0 can't be designated as
KM, because the enzyme doesn't follow the hyperbolic Michaelis-Menten relationship. In the
sigmoidal plot, the substrate concentration at half maximal velocity is designated as [S] 0. 5 or K0. 5
.

Figure 5: The sigmoid curve for

a
homotropic allosteric enzyme
Homotropic allosteric enzymes generally are multi-subunit proteins and the same binding
site on each subunit functions as both the active site and the regulatory site. The substrate acts as

a positive modulator as binding of one molecule of substrate to one binding site alters the
enzyme's conformation and enhances the binding of subsequent substrate molecules. So this
results in the sigmoid change in V0, with increasing substrate concentration. Sigmoid kinetics
involves small changes in the concentration of a modulator can be associated with large changes
in activity. The sigmoid curve for a homotropic enzyme (figure-5), shows a relatively small
increase in the substrate concentration, [S] causes a comparatively large increase in V0.
In heterotropic allosteric enzymes, the modulators are metabolites other than the normal
substrate, and therefore the substrate-saturation curve changes according to the modulator. Some
heterotropic allosteric enzymes have activating modulators, while some have inhibitory
modulators and some have both inhibitory and activating modulators. An activator or positive
modulator may cause the curve to become more nearly hyperbolic, with a decrease in K 0. 5 but no
change in Vmax, resulting in an increased reaction velocity at a fixed substrate concentration
(Figure-6, upper curve). A negative modulator or an inhibitor may produce a more sigmoid
substrate-saturation curve (Figure-6, lower curve), with an increase in K0. 5.

Figure 6: The effects of a positive modulator and a

negative
modulator on a heterotropic allosteric enzyme

Some heterotropic allosteric enzymes show a rare type of modulation, where Vmax is altered and
K0. 5 remains nearly constant (Figure-7).

Figure 7: A rare type of modulation

shown
by some heterotropic allsosteric
enzymes

The interaction between the different subunits can be expressed by a cooperativity coefficient n
(Hill coefficient). When n = 1 there is no interaction between the subunits, the enzyme show
Michaelis-Menten kinetics. The stronger the interactions between the subunits (more interactions
must be broken to allow the conformational changes due to substrate binding) the larger the Hill
coefficient.
A second parameter characterizes the shape of the curve. This is the K 0.5 value, the substrate
concentration at which the enzyme shows half of its maximum activity. When n = 1, K 0.5 is the
KM value.
The experimentally found "n" values vary between 1 and 4 for substrate induced conformational
changes. When there is a strong interaction (large "n") the substrate can act as a switch. In a
relative (compared to Michaelis-Menten kinetics) narrow substrate concentration range, the
enzyme switches from a low activity to a high activity. It should be realized that all or nothing
behaviour can not be achieved by allosteric regulation but only by (reversible) covalent
modification. Covalent modification can switch an enzyme from fully active to completely
inactive.
Hemoglobin, though not an enzyme, is the canonical example of an allosteric protein molecule and one of the earliest to have its crystal structure solved (by Max Perutz). More recently, the E.
coli enzyme aspartate carbamoyltransferase (ATCase) has become another good example of
allosteric regulation.
The kinetic properties of allosteric enzymes are often explained in terms of a conformational
change between a low-activity, low-affinity "tense" or T state and a high-activity, high-affinity
"relaxed" or R state. These structurally distinct enzyme forms have been shown to exist in
several known allosteric enzymes.
However the molecular basis for conversion between the two states is not well understood. Two
main models have been proposed to describe this mechanism: the "concerted model" of Monod,
Wyman, and Changeux, and the "sequential model" of Koshland, Nemethy, and Filmer.
In the concerted model, the protein is thought to have two all-or-none global states. This model

is supported by positive cooperativity where binding of one ligand increases the ability of the
enzyme to bind to more ligands. The model is not supported by negative cooperativity where
losing one ligand make it easier for the enzyme to lose more.
In the sequential model there are many different global conformational/energy states. Binding of
one ligand changes the enzyme so it can bind more ligands more easily i.e. every time it binds a
ligand it wants to bind another one.
Neither model fully explains allosteric binding, however. The recent combined use of physical
techniques (for example, x-ray crystallography and solution small angle x-ray scattering or
SAXS) and genetic techniques (site-directed mutagenesis or SDM) may improve our
understanding of allostery.
Models of allosteric regulation
The sigmoidal dependence of V0 on [S] reflects subunit co-operativity, has inspired two models
to explain these cooperative interactions. Most allosteric effects can be explained by the
concerted MWC model put forth by Monod, Wyman, and Changeux, or by the sequential model
described by Koshland, Nemethy, and Filmer. Both postulate that enzyme subunits exist in one
of two conformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more
readily than those in the tense state. The two models (figure-9) differ most in their assumptions
about subunit interaction and the pre-existence of both states.
The concerted model
The concerted model of allostery, postulates that enzyme subunits are connected in such a way
that a conformational change in one subunit is necessarily conferred to all other subunits or in
other words, all subunits must be in the same conformation. As mentioned earlier that allosteric
enzymes can exist in two states, i.e., relaxed (R state) and tight (T state). In this two-state model,
all the subunits of an oligomer must be in the same state (they all change together) and is
therefore termed the concerted model . T state predominates in the absence of substrate S and S
bind more tighter to R than T. The T and R state equilibrium depends upon the concentration of
the substrate. At high substrate concentration, more enzymes are found in the R state, whereas at
low substrate concentration, the enzymes are found in the T state. The equilibrium can be shifted
to the R or T state through the binding of the allosteric effector (activator or inhibitor) to the
allosteric site. Activator and inhibitor bind to R and T states respectively (Figure-8). In this
symmetry model, binding of ligand to one subunit always assists the binding of the same ligand
to the next subunit, i.e, only positive co-operativity is possible here. Heterotropic interactions
could either be positive or negative.

Figure 8: The concerted model for allosteric regulation

The sequential model for allosteric regulation

The sequential model (figure-9) of allosteric regulation holds that subunits are not connected in
such a way that a conformational change in one subunit induces a similar change in the other
subunits. Thus, all enzyme subunits do not necessitate the same conformation. Subunits may
undergo individual sequential changes in conformation. Subunits can interact in different
conformations. The sequential model says that molecules of substrate bind via an induced-fit
hypothesis. When a subunit randomly collides with a molecule of substrate, the active site, in
essence, forms a glove around its substrate. While such an induced fit of the substrate and a
subunit, cause a conformation change (Figure-9) in the subunit, converting it from the tensed
state to relaxed state (T form to R form), and increasing the sites available to the substrate. The
alteration of conformation of one subunit by substrate binding is transmitted to other subunits by
subunit intreractions, and symmetry needn't be conserved. Change induced by binding of
substrate to one subunit can increase or decrease substrate binding to other subunits, i.e., the
ligand-induced conformational change in one subunit can affect the adjoining subunit. That
means both positive and negative homotropic interactions are possible in this model.
Heterotropic interactions could either be positive or negative.

Figure 9: The sequential model for allosteric

regulation

LECTURE SEVEN

ZYMOGENS
Digestive enzymes are released in inactive forms called zymogens. This is
necessary to prevent the digestive enzymes from digesting the cells that produce
them. In a zymogen, a peptide blocks the active site of the enzyme. Cleaving off
this peptide activates the enzyme.
Activation of pepsinogen in the stomach

The peptidase in the stomach is pepsin. Pepsin works optimally in the acidic
environment of the stomach, being active at pH 2-3, but becoming inactivated
when the pH is above 5. The chief cells at the base of the gastric glands secrete the
zymogen, which is called pepsinogen. Activation of pepsinogen starts
whenhydrocholoric acid (HCl), which is secreted by the parietal cells partially
activates pepsinogen (pepsinogen* in figure). This partially active enzyme then
cleaves the peptide from other pepsinogen molecules to form active pepsin.

Activation of pancreatic zymogens in the small intestine

Pancreatic zymogens are normally only activated after they reach the small
intestine. A brush border enzyme, enterokinase, cleaves a peptide
from trypsinogen, forming the active enzyme trypsin. Trypsin then activates the
other enzymes. ("Brush border" is another term for themicrovilli at the apical
surface of enterocytes, where brush border enzymes are located).
A dangerous situation occurs if there is inappropriate formation of trypsin in the
pancreas. This can cause pancreatitis, where trypsin digests pancreatic tissue and
triggers an inflammatory response. Acinar cells synthesize and secrete a trypsin
inhibitor that acts as a safeguard against trypsin activation within the pancreas.
Another protective mechanism is that trypsin has a mechanisms of autolysis (selfdigestion). Genetic mutations that decrease the activity of the pancreatic trypsin
inhibitor increase the risk for pancreatitis, as do mutations that affect trypsinogen
so that it is more likely to become prematurely activated or is resistant to autolysis.
A further important mechanism to protect against pancreatitis is fluid secretion by
duct cells to flush zymogens (or active enzymes) out of the pancreas and into the
duodenum. Blockage of the pancreatic duct (for instance, by a gallstone) will
prevent flow out of the pancreas and can be a cause of acute pancreatitis. Fluid
secretion in the pancreas depends upon the chloride channel CFTR (as does fluid
secretion in the lungs and small intestine). Patients with mutations in the CFTR
gene (which causes cystic fibrosis) have an increased risk for the development of
pancreatitis.
Two other factors that increase the risk for the development of pancreatitis are
excessive alcohol consumption and hyperlipidemia. Alcohol and fatty acids cause
inappropriate intracellular activation of trypsin via mechanisms that are still being

elucidated.

LECTURE EIGHT

DIGESTIVE ENZYMES
Digestive enzymes all belong to the hydrolase class, and their action is one of splitting
up large food molecules into their building block components. Another
unique property is that they are extracellular enzymes that mix with food as it passes
through the gut. The majority of other enzymes function within the cytoplasm of
the cell.
The chemical digestion of food is dependent on a whole range of hydrolase enzymes
produced by the cells lining the gut as well as associated organs such as the pancreas.
The end goal is to break large food molecules into very much smaller building block
units. These can then be readily and rapidly absorbed through the gut wall and into the
bloodstream for transport to the liver and from there to other parts of the body.
The main enzyme-producing structures of the human digestive system are the salivary
glands, stomach, pancreas, liver and small intestine.
Digestive juices and
enzymes

Substance digested

Saliva
Amylase

Starch

Gastric juice
Protease (pepsin) and
hydrochloric acid

Proteins

Pancreatic juice
Proteases (trypsin)
Lipases
Amylase
Intestinal enzymes
Peptidases
Sucrase
Lactase
Maltase
Bile from the liver
Bile salts

Product formed

Maltose
Partly digested proteins
Proteins
Fats emulsified by bile
Starch

Peptides and amino acids

Fatty acids and glycerol
Maltose

Peptides
Sucrose (sugar)
Lactose (milk sugar)
Maltose

Amino
acids
Glucose and fructose
Glucose and galactose
Glucose

Fats
globules

Fat droplets

The following pathway summarises how starch present in a food like bread is broken
down chemically into glucose, which can then be absorbed through the intestinal wall
and into the bloodstream for transport to the liver and from there to other parts of the
body.

Mouth and duodenum

Starch hydrolysed into maltose through the action of the enzyme amylase.

Jejunum
Maltose hydrolysed into glucose through the action of the enzyme maltase.

LECTURE NINE
VITAMINS AND COENZYMES

COFACTORS AND COENZYMES

COFACTORS
Some enzymes do not need any additional components to show full activity. However, others
require non-protein molecules called cofactors to be bound for activity. Cofactors can be either
inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds (e.g., flavin and
heme). Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme,
or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes
include NADH, NADPH and adenosine triphosphate. These molecules transfer chemical groups
between enzymes.
An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the
ribbon diagram above with a zinc cofactor bound as part of its active site. These tightly bound
molecules are usually found in the active site and are involved in catalysis. For example, flavin
and heme cofactors are often involved in redox reactions.
Enzymes that require a cofactor but do not have one bound are called apoenzymes or
apoproteins. An apoenzyme together with its cofactor(s) is called a holoenzyme (this is the active
form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound.
However, organic prosthetic groups can be covalently bound (e.g., biotin in the enzyme pyruvate
carboxylase). The term "holoenzyme" can also be applied to enzymes that contain multiple
protein subunits, such as the DNA polymerases; here the holoenzyme is the complete complex
containing all the subunits needed for activity.

COENZYMES
Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme.
Tightly bound coenzymes can be called allosteric groups. Coenzymes transport chemical groups
from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid
are vitamins (compounds which cannot be synthesized by the body and must be acquired from
the diet). The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+,
the phosphate group carried by adenosine triphosphate, the acetyl group carried by coenzyme A,
formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by Sadenosylmethionine.
Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to
consider coenzymes to be a special class of substrates, or second substrates, which are common
to many different enzymes. For example, about 700 enzymes are known to use the coenzyme
NADH.
Coenzymes are usually continuously regenerated and their concentrations maintained at a steady
level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway
and S-adenosylmethionine by methionine adenosyltransferase. This continuous regeneration
means that even small amounts of coenzymes are used very intensively. For example, the human body
turns over its own weight in ATP each day.

TABLE OF CONTENTS
1 Chapter One: Steady State Enzyme Kinetics.
2 Chapter Two: Transient State Kinetic Methods.
3 Chapter Three: Chemistry of Enzyme Catalysis
4 Chapter Four: Regulatory Enzymes
5 Chapter Five: Molecular Models for Allosterism.
6 Chapter Six: Multi-enzyme Complexes,
7 Chapter Seven: Enzyme Assays
8 Chapter Eight: Criteria for Determining Purity of Enzymes
9 ChapterNine: Enzyme Reconstitution.
10 Chapter Ten:Regulation of Enzyme Activity and Synthesis

CHAPTER ONE
THE STEADY STATE KINETICS
WHAT IS A STEADY STATE?

In chemistry, a steady state is a situation in which all state variables are constant in spite
of ongoing processes that strive to change them. For an entire system to be at steady state, i.e. for
all state variables of a system to be constant, there must be a flow through the system (compare
mass balance). A simple example of such a system is the case of a bathtub with the tap
running but with the drain unplugged: after a certain time, the water flows in and out at the same
rate, so the water level (the state variable Volume) stabilizes and the system is in a steady state.
The steady state concept is different from chemical equilibrium. Although both may create a
situation where a concentration does not change, in a system at chemical equilibrium, the net
reaction rate is zero (products transform into reactants at the same rate as reactants transform
into products), while no such limitation exists in the steady state concept. Indeed, there does not
have to be a reaction at all for a steady state to develop.
The term steady state is also used to describe a situation where some, but not all, of the state
variables of a system are constant. For such a steady state to develop, the system does not have to
be a flow system. Therefore such a steady state can develop in a closed system where a series of
chemical reactions take place. Literature in chemical kinetics usually refers to this case,
calling it steady state approximation.
In simple systems the steady state is approached by state variables gradually decreasing or
increasing until they reach their steady state value. In more complex systems state variable might
fluctuate around the theoretical steady state either forever (a limit cycle) or gradually coming
closer and closer. It theoretically takes an infinite time to reach steady state, just as it takes an
infinite time to reach chemical equilibrium.
Both concepts are, however, frequently used approximations because of the substantial
mathematical simplifications these concepts offer. Whether or not these concepts can be used
depends on the error the underlying assumptions introduce. So, even though a steady state, from
a theoretical point of view, requires constant drivers (e.g. constant inflow rate and constant
concentrations in the inflow), the error introduced by assuming steady state for a system with
non-constant drivers may be negligible if the steady state is approached fast enough (relatively
speaking).
Steady state approximation in chemical kinetics
The steady state approximation, occasionally called the stationary-state approximation,
involves setting the rate of change of a reaction intermediate in a reaction mechanism
equal to zero.
It is important to note that steady state approximation does not assume the reaction
intermediate concentration to be constant (and therefore its time derivative being zero), it
assumes that the variation in the concentration of the intermediate is almost zero: the
concentration of the intermediate is very low, so even a big relative variation in its concentration
is small, if considered quantitatively.

Its use facilitates the resolution of the differential equations that arise from rate
equations, which lack an analytical solution for most mechanisms beyond the most simple
ones. The steady state approximation is applied, for example in Michaelis-Menten kinetics.
REFERENCES
1

^IUPAC Gold Book definition of steady state

^ P. W. Atkins "Physical Chemistry" For a complete derivation of the exact and

approximate solution and a comparison between them

ENZYME KINETICS
THE ENZYME SUBSTRATE COMPLEX
A theory to explain the catalytic action of enzymes was proposed by the Swedish Chemist
Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some
intermediate substance which is known as the enzyme substrate complex. The reaction can be
represented as:
E+S

ES

E+P

This indicates that the enzyme first unites in some way with the substrate and then returns to its
original form after the reaction is concluded.
Enzyme kinetics is a branch of enzymology that deals with the factors affecting the rate of
enzyme catalyzed reactions. The most important factors involved among others:
(1) The enzyme concentration
(2) Ligand concentration (substrate, products, inhibitors and activators).
(3) pH
(4) Ionic strength
(5) Temperature
During deacylation, the OH ion simultaneously attacks a carbonyl carbon atom of the acyl group
that is attached to Ser-195, His-57,then donate a proton to the oxygen atom of Ser-195, which
result in the release of the acid component of substrate. The acid component then diffuse away
and the enzyme is ready for another round of catalysis.

The Steady State Kinetics

The kinetics of enzymecatalysed reactions can be analysed in terms of steady state models if the
substrate concentrations are more than an order of magnitude higher than the enzyme level. Such
analysis yields Michaelis constants, maximum velocities and useful information on reaction
mechanisms. Thus when there are two or more substrates, the number of terms in the
denominator of the rate equation tells the type of mechanism. A sequential mechanism has a
constant term plus ones in the concentration of each substrate and one including both
concentrations. A ping pong mechanism has no constant term in the denominator. An ordered
sequential mechanism where the binding of the first substrate is at equilibrium lacks a
denominator term in the concentration of the second substrate. Other types of steady state studies

include the use of inhibitors, determination of pH profiles and the effect of isotopic substitutions
on the rates.
Key Concepts:
As long as substrate concentrations are more than an order of magnitude greater than the
enzyme level, the reaction will be in a steady state.

Steady state rate equations are simple ratios of a numerator that is a function of substrate
concentrations and a maximum velocity divided by a denominator that represents the
distribution of the enzyme among all possible forms that are present in the steady state.

Steady state kinetic studies enable one to distinguish kinetic mechanisms and can also
give information on rate limiting steps and in favorable cases, chemical mechanism.

Keywords: enzyme kinetics; steady state; initial velocities; rate equations; kinetic constants
In most cases, an enzyme converts one chemical (the substrate), into another (the product). A
graph of product concentration vs. time follows three phases as shown in the following graph.

The Michaelis- Menten Kinetics

The primary function of enzymes is to enhance rates of reactions so that they are compatible with
the needs of the organism. To know about how enzymesfunction, a kinetic description of their
activity is needed. The study of chemical reactions catalyzed by enzymes is known as enzyme
kinetics.
As enzyme-catalysed reactions are saturable, their rate of catalysis does not show a linear
response to increasing substrate. If the initial rate of the reaction is measured over a range of

substrate concentrations (denoted as [S]), the reaction rate (V0) increases as [S] increases, as
shown in figure-1.

Figure 1: Saturation curve for an enzyme

which obeys Michaelis-Menten kinetics

However, as [S] gets higher, the enzyme becomes saturated with substrate and the rate
reaches Vmax, the enzyme's maximum rate. In 1913, Leonor Michaelis and Maud Menten
proposed a simple model to account for these kinetic characteristics. In this model, a specific ES
complex is a necessary intermediate in catalysis. Consider an enzyme that catalyzes the S to P
by the following pathway:

An enzyme E combines with substrate S to form an ES complex, with a rate constant k1. The
ES complex can dissociate to E and S with a rate constant k-1, or it can proceed to form product
P, with a rate constant k2. It can also be assumed that almost none of the product reverts to the
initial substrate, a condition that holds in the initial stage of a reaction before the concentration of
product is appreciable.
The catalytic rate is equal to the product of the concentration of the ES complex and k2.
V0 = k2 [ES]-------------Eqn (1)
The rate of formation of ES is given by:
[ES] = k1 [E] [S]-----------Eqn (2)

The rate of breakdown of ES is given by:

[ES] = (k-1 + k2) [ES]------------Eqn (3)
Considering a steady state, [ES], stay the same even if the concentrations of starting materials
and products are changing. This occurs when the rates of formation and breakdown of the ES
complex are equal. Therefore equating equations 2 and 3,
k1 [E] [S] = (k-1 + k2) [ES], which can be modified to
( [E] [S] ) / [ES] =

( k-1 + k2) / k1--------------Eqn (4)

Equation 4 can be simplified by defining a new constant, KM, called the Michaelis constant:
KM = ( k-1 + k2) / k1----------------------Eqn (5)
KM is an important characteristic of enzyme-substrate interactions and is independent of enzyme
and substrate concentrations.
Considering equations 4 and 5, [ES] can be determined, which is
[ES] = ( [E] [S] ) / KM -------------------------Eqn (6)
The concentration of uncombined substrate [S] is very nearly equal to the total substrate
concentration, provided that the concentration of enzyme is much lower than that of substrate.
The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E]T
minus the concentration of the ES complex. Therefore
[E] = [E]T [ES] ------------------------- eqn (7)
Equation 6 can be written as
[ES] = ( ( [E]T [ES] ) [S] ) / KM -------------------------Eqn (8)
Solving equation 8,
[ES] =

( ( [E]T [S] ) / KM ) / (1 + [S] / KM ) -------------------Eqn (9)

Equation 9 can be modified as

[ES] = [E]T ( [S] / [S] + KM )
Substituting the value of [ES] from equation 9 in equation 1,
V0 = k2 [E]T ( [S] / [S] + KM ) -------------------Eqn (10)

The maximal rate, Vmax, is attained when the catalytic sites on the enzyme are saturated with
substrate, that means when
[ES] = [E]T.
Therefore,
Vmax = k2 [E]T --------------------------Eqn (11)
Putting the value of Vmax from equation 11 in equation 10, we get
V0 = Vmax ( [S] / [S] + KM ) ------------------- The Michaelis Menten equation.
The equation states that, at very low substrate concentration, when [S] is much less than KM,
Then,V0 = (Vmax / KM) [S]; that is, the rate is directly proportional to the substrate concentration.
At high substrate concentration, when [S] is much greater than KM, Then V0 = Vmax ; that is, the
rate is maximal, independent of substrate concentration.

CHAPTER TWO

TRANSCIENT (PRE-STEADY) STATE KINETICS

The pre-steady-state or transient phase in enzyme-catalysed reactions occupies very short
periods (usually fractions of a second) and very low product concentrations. Special techniques
therefore have to be used. For reactions that are not too fast the stopped-flow technique, in which
the enzyme and reactants are rapidly mixed and the flow stopped is commonly used.
For reactions that have to be studied over periods of less than 1 ms relaxation techniques are
used. In these techniques the system is disturbed, usually but not necessarily from a state of
equilibrium, after which it relaxes to equilibrium or a new steady state. In the temperature-jump
(T jump) technique the temperature is increased rapidly and the system relaxes to a new state of
equilibrium or a new steady state at the final temperature. In the pressure-jump technique the
pressure is rapidly changed.
The relaxation time of a reaction is the time it takes for the extent of reaction to change by a
proportion (1 - e-1) of the total change during the relaxation process (e = 2.71828 . . . is the base
of natural logarithms). For composite mechanisms, such as those that occur with enzymecatalysed reactions, relaxation experiments usually reveal more than one relaxation time. These
relaxation times can be related to the rate constants of the elementary steps in the mechanism,
but the relationships are usually complicated.

Pre-steady-state kinetics

Pre-steady state progress curve, showing the burst phase of an enzyme reaction.
In the first moment after an enzyme is mixed with substrate, no product has been formed and no
intermediates exist. The study of the next few milliseconds of the reaction is called Presteady-state kinetics also referred to as Burst kinetics. Pre-steady-state kinetics is therefore
concerned with the formation and consumption of enzymesubstrate intermediates (such as ES
or E*) until their steady-state concentrations are reached.

This approach was first applied to the hydrolysis reaction catalysed by chymotrypsin.[33]
Often, the detection of an intermediate is a vital piece of evidence in investigations of what
mechanism an enzyme follows. For example, in the pingpong mechanisms that are shown
above, rapid kinetic measurements can follow the release of product P and measure the formation
of the modified enzyme intermediate E*.[34] In the case of chymotrypsin, this intermediate is
formed by an attack on the substrate by the nucleophilic serine in the active site and the
formation of the acyl-enzyme intermediate.
In the figure to the right, the enzyme produces E* rapidly in the first few seconds of the reaction.
The rate then slows as steady state is reached. This rapid burst phase of the reaction measures a
single turnover of the enzyme. Consequently, the amount of product released in this burst, shown
as the intercept on the y-axis of the graph, also gives the amount of functional enzyme which is
present in the assay.[35]

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry
(ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic
reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this
approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or betaxylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme
intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5dinitrophenyl beta-D-xylobioside was detected by time-resolved ESI-MS. The low concentration
of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By
contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the
same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the
formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic
data are in agreement with those determined by monitoring the release of 2,5-dinitrophenol with
stopped-flow W-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to
accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of
identifying transient enzyme intermediates by their mass.

THE TRANSCIENT STATE

PHASE 1
At very early time points, the rate of product accumulation increases over time. Special
techniques are needed to study the early kinetics of enzyme action; since this transient phase
usually lasts less than a second (the figure greatly exaggerates the first phase).

PHASE 2
For an extended period of time, the product concentration increases linearly with time.

At later times, the substrate is depleted, so the curve starts to level off. Eventually the
concentration of product reaches a plateau and doesn't change with time.
It is difficult to fit a curve to a graph of product as a function of time, even if you use a simplified
model that ignores the transient phase and assumes that the reaction is irreversible. The model
simply cannot be solved to an equation that expresses product concentration as a function of
time. To fit these kind of data (called an enzyme progress curve) you need to use a program that
can fit data to a model defined by differential equations or by an implicit equation. Prism cannot
do this.
Rather than fit the enzyme progress curve, most analyses of enzyme kinetics fit the initial
velocity of the enzyme reaction as a function of substrate concentration. The velocity of the
enzyme reaction is the slope of the linear phase, expressed as amount of product formed per
time. If the initial transient phase is very short, you can simply measure product formed at a
single time, and define the velocity to be the concentration divided by the time interval.
This lecture considers data collected only in the second phase. The terminology describing these
phases can be confusing. The second phase is often called the "initial rate", ignoring the short
transient phase that precedes it. It is also called "steady state", because the concentration of
enzyme-substrate complex doesn't change. However, the concentration of product accumulates,
so the system is not truly at steady state until, much later, the concentration of product truly
doesn't change over time.
Enzyme velocity as a function of substrate concentration
If you measure enzyme velocity at many different concentrations of substrate, the graph
generally looks like this:

Enzyme velocity as a function of substrate concentration often follows the Michaelis-Menten

equation:

Vmax is the limiting velocity as substrate concentrations get very large. Vmax (and V) are
expressed in units of product formed per time. If you know the molar concentration of enzyme,
you can divide the observed velocity by the concentration of enzyme sites in the assay, and
express Vmax as units of moles of product formed per second per mole of enzyme sites. This is
the turnover number, the number of molecules of substrate converted to product by one enzyme
site per second. In defining enzyme concentration, distinguish the concentration of enzyme
molecules and concentration of enzyme sites (if the enzyme is a dimer with two active sites, the
molar concentration of sites is twice the molar concentration of enzyme).
KM is expressed in units of concentration, usually in Molar units. KM is the concentration of
substrate that leads to half-maximal velocity. To prove this, set [S] equal to KM in the equation
above. Cancel terms and you'll see that V=Vmax/2.
The meaning of KM
To understand the meaning of Km, you need to have a model of enzyme action. The simplest
model is the classic model of Michaelis and Menten, which has proven useful with many kinds
of enzymes.

The substrate (S) binds reversibly to the enzyme (E) in the first reaction. In most cases, you can't
measure this step. What you measure is production of product (P), created by the second
reaction.
From the model, we want to derive an equation that describes the rate of enzyme activity
(amount of product formed per time interval) as a function of substrate concentration.
The rate of product formation equals the rate at which ES turns into E+P, which equals k2 times
[ES]. This equation isn't helpful, because we don't know ES. We need to solve for ES in terms of
the other quantities. This calculation can be greatly simplified by making two reasonable
assumptions. First, we assume that the concentration of ES is steady during the time intervals
used for enzyme kinetic work. That means that the rate of ES formation, equals the rate of ES
dissociation (either back to E+S or forward to E+P). Second, we assume that the reverse reaction
(formation of ES from E+P) is negligible, because we are working at early time points where the
concentration of product is very low.

We also know that the total concentration of enzyme, Etotal, equals ES plus E. So the equation
can be rewritten.

Solving for ES:

The velocity of the enzyme reaction therefore is:

Finally, define Vmax (the velocity at maximal concentrations of substrate) as k2 times Etotal,
and KM, the Michaelis-Menten constant, as (k2+k-1)/k1. Substituting:

Note that Km is not a binding constant that measures the strength of binding between the enzyme
and substrate. Its value includes the affinity of substrate for enzyme, but also the rate at which
the substrate bound to the enzyme is converted to product. Only if k2 is much smaller than k-1
will KM equal a binding affinity.The Michaelis-Menten model is too simple for many purposes.
The Briggs-Haldane model has proven more useful:

Under the Briggs-Haldane model, the graph of enzyme velocity vs. substrate looks the same as
under the Michaelis-Menten model, but KM is defined as a combination of all five of the rate
constants in the model.
Assumptions of enzyme kinetic analyses
Standard analyses of enzyme kinetics (the only kind discussed here) assume:

The production of product is linear with time during the time interval used.
The concentration of substrate vastly exceeds the concentration of enzyme. This means that
the free concentration of substrate is very close to the concentration you added, and that

substrate concentration is constant throughout the assay.

A single enzyme forms the product.

There is negligible spontaneous creation of product without enzyme

REFERENCES
RG Duggleby, "Analysis of Enzyme Reaction Progress Curves by Nonlinear Regression",
Methods in Enzymology, 249: page 60-, 1995.

CHAPTER THREE
CHEMISTRY OF ENZYME CATALYSIS
Enzyme catalysis
Enzymes act by lowering the activation energy. They do not change the equilibrium of the reactions. Enzyme binds with the
substrate to form an unstable complex which breaks up into products liberating enzymes. Lock and key theory and induced
fit theory explain the enzyme-substrate complex formation. Small region on the enzyme where the substrate binds and
catalysis takes place is called an active site. Enzymes are highly specific in their action. Three types of enzyme specificity
are well recognized: They are stereo specificity, reaction specificity and substrate specificity. There are different
mechanisms to explain enzyme catalysis. Different mechanisms are: acid base catalysis, covalent catalysis and metal ion
catalysis and transition state stabilization.

CHAPTERFOUR
REGULATORY ENZYMES

REGULATORYENZYME
A regulatory enzyme is an enzyme in a biochemicalpathway which, through its responses to the
presence of certain other biomolecules, regulates the pathway's activity. This is usually done for
pathways whose products may be needed in different amounts at different times, such as
hormone production. Regulatory enzymes exist at high concentrations (low Vmax) so its activity
can be increased or decreased with changes in substrate concentrations.
Regulatory enzymes exhibit increased or decreased catalytic activity in response to
certain signals. By the action of such regulatory enzymes, the rate of each metabolic sequence is
constantly adjusted to meet changes in the cell's demands for energy and for biomolecules
required in cell growth and repair. In most multienzyme systems the first enzyme of the sequence
is a regulatory enzyme. Catalyzing even the first few reactions of a pathway that leads to an
unneeded product diverts energy and metabolites from more important processes. An excellent
place to regulate a metabolic pathway, therefore, is at the point of commitment to the pathway.
The other enzymes in the sequence are usually present in amounts providing a large excess of
catalytic activity; they can promote their reactions only as fast as their substrates are made
available from preceding reactions.
The activity of regulatory enzymes is modulated through various types of signal molecules,
which are generally small metabolites or cofactors. There are two major classes of regulatory
enzymes in metabolic pathways: Allosteric enzymes and covalently modulated enzymes.

ALLOSTERIC ENZYMES:Allosteric enzymes have two binding sites; one for the
substrate and the other for small molecules called effectors which modulates the enzymes
activity. Effectors are non-covalently linked to the enzyme at its allosteric site (site of enzyme
where the effector binds) and its interaction with the enzyme is reversible. Based on modulation,
allosteric enzymes can be grouped into two groups: 1.Homotropic allosteric enzyme and
2.Heterotropic allosteric enzymes. In the homotropic allosteric enzyme both the substrate and the
effector plays part in the modulation of the enzyme, which in turn affects the enzyme catalytic
activity. In the heterotropic form it is only the effector that performs the role of modulation.At
this junction the allosteric site of the enzyme could range from one(monovalent) or from two and
above (polyvalent). Usually regulatory enzyme starts the catalysis of multienzyme reaction e.g.
gycolysis, fat synthesis e.t.c.An example of an allosteric enzyme is Aspartate transcarbamoylase.
This enzyme catalyzes the synthesis of pyrimidine nucleotides.

ALLOSTERIC ENZYMES
Allosteric enzymes function through reversible, noncovalent binding of a regulatory
metabolite called a modulator. The term allosteric derives from Greek allos, "other," and stereos,
"solid" or "shape." Allosteric enzymes are those having "other shapes" or conformations induced
by the binding of modulators.

To a degree, allosteric (noncovalent) regulation may permit fine-tuning of metabolic pathways

that are required continuously but at different levels of activity as cellular conditions change.
ALLOSTERIC ENZYMES ARE REGULATED BY NONCOVALENT BINDING OF
MODULATORS
In some multienzyme systems the regulatory enzyme is specifically inhibited by the end product
of the pathway, whenever the end product increases in excess of the cell's needs. When the
regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced rates because
their substrates are depleted by mass action. The rate of production of the pathway's end product
is thereby brought into balance with the cell's needs. This type of regulation is called
feedback inhibition. Buildup of the pathway's end product ultimately slows the entire
pathway.
One of the first discovered examples of such
allosteric feedback inhibition was the bacterial
enzyme system that catalyzes the conversion of
L-threonine into r.-isoleucine (Fig 1).
In this system, the first enzyme, threonine
dehydratase, is inhibited by isoleucine, the
product of the last reaction of the series.
Isoleucine is quite specific as an inhibitor. No
other intermediate in this sequence of reactions
inhibits threonine dehydratase, nor is any other
enzyme in the sequence inhibited by
isoleucine. Isoleucine binds not to the active
site, but to another specific site on the enzyme
molecule, the regulatory site. This binding is
noncovalent and thus readily reversible; if the
isoleucine concentration decreases, the rate of
threonine dehydratase activity increases. Thus
threonine dehydratase activity responds rapidly
and reversibly to fluctuations in the
concentration of isoleucine in the cell.

Figure 1:Feedback inhibition of the conversion of L-threonine into L-isoleucine, catalyzed by a sequence of five
enzymes (El to E5). Threonine dehydratase (El) is specifically inhibited allosterically by L-isoleucine, the end
product of the sequence, but not by any of the four intermediates (A to D). Such inhibition is indicated by the dashed
feedback line and the @ symbol at the threonine dehydratase reaction arrow

ALLOSTERIC ENZYMES ARE EXCEPTIONS TO MANY GENERAL RULES

The modulators for allosteric enzymes may be either inhibitory or stimulatory. An activator is
often the substrate itself, and regulatory enzymes for which substrate and modulator are identical

are called homotropic.When the modulator is a molecule other than the substrate the
enzyme is heterotropic. Some enzymes have two or more modulators.
As already noted, the properties of allosteric enzymes are
significantly different from those of simple nonregulatory
enzymes. Some of the differences are structural. In addition
to active or catalytic sites, allosteric enzymes generally have
one or more regulatory or allosteric sites for binding the
modulator (Fig 2). Just as an enzyme's active site is specific
for its substrate, the allosteric site is specific for its
modulator. Enzymes with several modulators generally have
different specific binding sites for each. In homotropic
enzymes the active site and regulatory site are the same.
Figure 2:Schematic model of the subunit interactions in an allosteric enzyme, and interactions with inhibitors and
activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different
subunits, the catalytic (C)and regulatory (R) subunits, respectively. Binding of the positive modulator (M) to its
specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change.
This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On
dislocation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.

Allosteric enzymes are also generally larger and more complex than simple enzymes. Most of
them have two or more polypeptide chains or subunits. Aspartate transcarbamoylase, which
catalyzes the first reaction in the biosynthesis of pyrimidine nucleotides, has 12 polypeptide
chains organized into catalytic and regulatory subunits. Figure 3 shows the quaternary structure
of this enzyme, deduced from x-ray analysis.

Figure 3: The three-dimensional subunit architecture of the regulatory enzyme aspartate transcarbamoylase; two
different views. This allosteric regulatory enzyme has two catalytic clusters, each with three catalytic polypeptide
chains, and three regulatory clusters, each with two regulatory polypeptide chains. The catalytic polypeptides in
each cluster are shown in shades of blue and purple. Binding sites for allosteric modulators are found on the
regulatory subunits (shown in white and red). Modulator binding produces large changes in enzyme conformation
and activity.

Other differences between nonregulated enzymes and allosteric enzymes involve kinetic
properties. Allosteric enzymes show relationships between V0 and [S] that differ from normal

Michaelis-Menten behavior. They do exhibit saturation with the substrate when [S] is sufficiently
high, but for some allosteric enzymes, when V0 is plotted against [S] (Fig 4) a sigmoid saturation
curve results, rather than the hyperbolic curve shown by nonregulatory enzymes. Although we
can find a value of [S] on the sigmoid saturation curve at which V 0 is half maximal, we cannot
refer to it with the designation Km because the enzyme does not follow the hyperbolic MichaelisMenten relationship. Instead the symbol [S]0.5 or K0.5 is often used to represent the substrate
concentration giving half maximal velocity of the reaction catalyzed by an allosteric enzyme (Fig
4).

Figure 4: Substrate-activity curves for representative allosteric enzymes. Three examples of complex responses
given by allosteric enzymes to their modulators. (a) The sigmoid curve given by a homotropic enzyme, in which the
substrate also serves as a positive (stimulatory) modulator. Note that a relatively small increase in [S] in the steep
part of the curve can cause a very large increase in V0. Note also the resemblance to the oxygen-saturation curve of
hemoglobin (see Fl 7-28). (b) The effects of a positive modulator
a negative modulator
, and no
modulator

on an allosteric enzyme in which K0.5 is modulated without a change in Vmax. (c) A less

common type of modulation, in which Vmax is modulated with K0.5 nearly constant.

Sigmoid kinetic behavior generally reflects cooperative interactions between multiple protein
subunits. In other words, changes in the structure of one subunit are translated into structural
changes in adjacent subunits, an effect that is mediated by noncovalent interactions at the
subunit-subunit interface. The principles are similar to those discussed for cooperativity in
oxygen binding to the nonenzyme protein hemoglobin. Homotropic allosteric enzymes generally
have multiple subunits. In many cases the same binding site on each subunit functions as both
the active site and the regulatory site. The substrate can function as a positive modulator (an
activator) because the subunits act cooperatively. The binding of one molecule of the substrate to
one binding site alters the enzyme's conformation and greatly enhances the binding of
subsequent substrate molecules. This accounts for the sigmoid rather than hyperbolic increase in
V0 with increasing [S].

With heterotropic enzymes, in which the modulator is a metabolite other than the substrate itself,
it is difficult to generalize about the shape of the substrate-saturation curve. An activator may
cause the substrate-saturation curve to become more nearly hyperbolic, with a decrease in K 0.5
but no change in Vmax, thus resulting in an increased reaction velocity at a fixed substrate
concentration ( V0) is higher for any value of [S]) (Fig 4b). Other allosteric enzymes respond to
an activator by an increase in Vmax, with little change in K0.5 (Fig 4c). A negative modulator (an
inhibitor) may produce a more sigmoid substrate-saturation curve, with an increase in K 0.5 (Fig 4
b). Allosteric enzymes therefore show different kinds of responses in their substrate-activity
curves because some have inhibitory modulators, some have activating modulators and some
have both.
TWO MODELS EXPLAIN THE KINETIC BEHAVIOR OF ALLOSTERIC ENZYMES
The sigmoidal dependence of V0 on [S] reflects subunit cooperativity, and has inspired two
models to explain these cooperative interactions.
THE SYMMETRY MODEL: proposed by Jacques Monod and colleagues in 1965.An
allosteric enzyme can exist in only two conformations, active and inactive (Fig. 5a). All subunits
are in the active form or all are inactive. Every substrate molecule that binds increases the
probability of a transition from the inactive to the active state.
THE SEQUENTIAL MODEL: (Fig. 5b), proposed by Koshland in 1966. There are still two
conformations, but subunits can undergo the conformational change individually. Binding of
substrate increases the probability of the conformational change. A conformational change in one
subunit makes a similar change in an adjacent subunit, as well as the binding of a second
substrate molecule, more likely. There are more potential intermediate states in this model than
in the symmetry model. The two models are not mutually exclusive; the symmetry model may be
viewed as the "all-or-none" limiting case of the sequential model. The precise mechanism of
allosteric interaction has not been established. Different allosteric enzymes may have different
mechanisms for cooperative interactions.

Figure 5: Two general models for the interconversion of inactive and active forms of allosteric enzymes. Four
subunits are shown because the model was originally proposed for the oxygen-carrying protein hemoglobin. In the
symmetry, or all-ornone, model (a) all the subunits are postulated to be in the same conformation, either all
affinity or inactive) or all

(high affinity or active). Depending on the equilibrium, K l, between

and

(low
forms,

the binding of one or more substrate (S) molecules will pull the equilibrium toward the
form. Subunits with
bound S are shaded. A possible pathway is given by the gray shading. In the sequential model (b) each individual
subunit can be in either the
or
form. A very large number of conformations is thus possible, but the shaded
pathway (diagonal arrows) is the most probable route.

COVALENTLY MODULATED ENZYME:Here,active

and inactive forms of

the enzyme are interconverted by covalent modification of their structures which are catalysed
by other enzymes.A typical example is glycogen phosphorylase which occurs in two forms,
Phoshorylase A,the more active one and Phosphorylase B, the less active one

REGULATORY ENZYME

REGULATORY ENZYME
A highly specialized enzyme having a regulatory (controlling) function through its capacity to
undergo a change in its catalytic activity. There exist two major types of regulatory enzymes: (1)
covalently modulated enzymes, and (2) allosteric enzymes.
A regulatory enzyme is an enzyme in a biochemicalpathway which, through its responses to the
presence of certain other biomolecules, regulates the pathway's activity. This is usually done for
pathways whose products may be needed in different amounts at different times, such as

hormone production. Regulatory enzymes exist at high concentrations (low Vmax) so its activity
can be increased or decreased with changes in substrate concentrations.
Regulatory enzymes exhibit increased or decreased catalytic activity in response to
certain signals. By the action of such regulatory enzymes, the rate of each metabolic sequence is
constantly adjusted to meet changes in the cell's demands for energy and for biomolecules
required in cell growth and repair. In most multienzyme systems the first enzyme of the sequence
is a regulatory enzyme. Catalyzing even the first few reactions of a pathway that leads to an
unneeded product diverts energy and metabolites from more important processes. An excellent
place to regulate a metabolic pathway, therefore, is at the point of commitment to the pathway.
The other enzymes in the sequence are usually present in amounts providing a large excess of
catalytic activity; they can promote their reactions only as fast as their substrates are made
available from preceding reactions.
The activity of regulatory enzymes is modulated through various types of signal molecules,
which are generally small metabolites or cofactors. There are two major classes of regulatory
enzymes in metabolic pathways: Allosteric enzymes and covalently modulated enzymes.
FEEDBACK INHIBITION
If enzymes worked 100% of the time, then we would never be able to store up any molecules for
energy and building materials in cells. So enzyme activity must be regulated and therefore are
involved in regulatory pathways. Enzymes simply work by bumping into molecules and those
that interact with the enzymes active site will be changed or modified in the process. The trick
comes in regulating the enzyme to control the pathway. Often an enzyme activity will be blocked
by something that is produced within the pathway itself. If molecule A is converted to B and
molecule B blocks or slows the enzyme down, then the pathways progress will be slowed...the
pathway is therefore regulated. As molecule B is metabolically used up then the enzyme is less
inhibited and the rate the pathway operates increases until the concentration of B is again built
up, and so the cycle goes. This is called metabolic feedback and it is how we as living systems
regulate our metabolic pathways.
Metabolic reactions in the cell are controlled by regulating enzymes. Regulatory mechanisms
involve not only inhibitory effects, but activating effects as well. Regulatory control is achieved
by controlling a few key enzymes in a series of reactions which occur in sequential steps guided
by complex arrays of enzymes. The product of one step becomes the substrate for the next step
and so forth. The whole series can be regulated by controlling the first enzyme in the series. The
first enzyme may be called the regulatory or allosteric enzyme.
The allosteric enzyme may be controlled by a feedback or end product inhibition at an
allosteric site different from the active site. Allosteric means "other site" or "other structure". The
interaction of an inhibitor at an allosteric site changes the structure of the enzyme so that the
active site is also changed.
This means that the product of the last reaction inhibits the first enzyme in the series. If the final
product accumulates to an excess, it inhibits the first enzyme and "switches off" the reaction.
When the product molecule concentration is lowered, the inhibiting molecule is removed so that
the first enzyme is "switched on" again.
The substrate interacts with the enzyme-1 to form products. One of these products becomes the
substrate for the next enzyme, and continues through several more enzymes. When a higher
concentraion of final product begins to build up, it "feedsback" to the first enzyme and reacts at
the allosteric site. The enzyme now changes shape so that the substrate does not fit into the active
site, therefore no more reactions can take place.

The net result is that the final product or feedback inhibitor acts as the on-off switch for the first
enzyme. When the concentration of product is high the enzyme is "turned off". But when the
concentration of final product is low, the feedback inhibitor (final product) moves out of the
allosteric site in a reversible reaction to "turn on" the enzyme once again.

ALLOSTERIC ENZYMES: Allosteric enzymes have two binding sites; one for the
substrate and the other for small molecules called effectors which modulates the enzymes
activity. Effectors are non-covalently linked to the enzyme at its allosteric site (site of enzyme
where the effector binds) and its interaction with the enzyme is reversible. Based on modulation,
allosteric enzymes can be grouped into two groups: 1. Homotropic allosteric enzyme and 2.
Heterotropic allosteric enzymes. In the homotropic allosteric enzyme both the substrate and the
effector plays part in the modulation of the enzyme, which in turn affects the enzyme catalytic
activity. In the heterotropic form it is only the effector that performs the role of modulation. At
this junction the allosteric site of the enzyme could range from one (monovalent) or from two
and above (polyvalent). Usually regulatory enzyme starts the catalysis of multienzyme reaction
e.g. gycolysis, fat synthesis e.t.c. An example of an allosteric enzyme is Aspartate
transcarbamoylase. This enzyme catalyzes the synthesis of pyrimidine nucleotides.

ALLOSTERIC ENZYMES
Allosteric enzymes function through reversible, noncovalent binding of a regulatory
metabolite called a modulator. The term allosteric derives from Greek allos, "other," and stereos,
"solid" or "shape." Allosteric enzymes are those having "other shapes" or conformations induced
by the binding of modulators.
To a degree, allosteric (noncovalent) regulation may permit fine-tuning of metabolic pathways
that are required continuously but at different levels of activity as cellular conditions change.
ALLOSTERIC ENZYMES ARE REGULATED BY NONCOVALENT BINDING OF
MODULATORS
In some multienzyme systems the regulatory enzyme is specifically inhibited by the end product
of the pathway, whenever the end product increases in excess of the cell's needs. When the
regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced rates because
their substrates are depleted by mass action. The rate of production of the pathway's end product
is thereby brought into balance with the cell's needs. This type of regulation is called feedback
inhibition. Buildup of the pathway's end product ultimately slows the entire pathway.

One of the first discovered examples of such

allosteric feedback inhibition was the bacterial
enzyme system that catalyzes the conversion of
L-threonine into r.-isoleucine (Fig 1).
In this system, the first enzyme, threonine
dehydratase, is inhibited by isoleucine, the
product of the last reaction of the series.
Isoleucine is quite specific as an inhibitor. No
other intermediate in this sequence of reactions
inhibits threonine dehydratase, nor is any other
enzyme in the sequence inhibited by
isoleucine. Isoleucine binds not to the active
site, but to another specific site on the enzyme
molecule, the regulatory site. This binding is
noncovalent and thus readily reversible; if the
isoleucine concentration decreases, the rate of
threonine dehydratase activity increases. Thus
threonine dehydratase activity responds rapidly
and reversibly to fluctuations in the
concentration of isoleucine in the cell.

Figure 1:Feedback inhibition of the conversion of L-threonine into L-isoleucine, catalyzed by a sequence of five
enzymes (El to E5). Threonine dehydratase (El) is specifically inhibited allosterically by L-isoleucine, the end
product of the sequence, but not by any of the four intermediates (A to D). Such inhibition is indicated by the dashed
feedback line and the @ symbol at the threonine dehydratase reaction arrow

ALLOSTERIC ENZYMES ARE EXCEPTIONS TO MANY GENERAL RULES

The modulators for allosteric enzymes may be either inhibitory or stimulatory. An activator is
often the substrate itself, and regulatory enzymes for which substrate and modulator are identical
are called homotropic. When the modulator is a molecule other than the substrate the enzyme is
heterotropic. Some enzymes have two or more modulators.

As already noted, the properties of allosteric enzymes are

significantly different from those of simple nonregulatory
enzymes. Some of the differences are structural. In addition
to active or catalytic sites, allosteric enzymes generally have
one or more regulatory or allosteric sites for binding the
modulator (Fig 2). Just as an enzyme's active site is specific
for its substrate, the allosteric site is specific for its
modulator. Enzymes with several modulators generally have
different specific binding sites for each. In homotropic
enzymes the active site and regulatory site are the same.
Figure 2: Schematic model of the subunit interactions in an allosteric enzyme, and interactions with inhibitors and
activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different
subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding of the positive modulator (M) to its
specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change.
This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On
dislocation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.

Allosteric enzymes are also generally larger and more complex than simple enzymes. Most of
them have two or more polypeptide chains or subunits. Aspartate transcarbamoylase, which
catalyzes the first reaction in the biosynthesis of pyrimidine nucleotides, has 12 polypeptide
chains organized into catalytic and regulatory subunits. Figure 3 shows the quaternary structure
of this enzyme, deduced from x-ray analysis.

Figure 3: The three-dimensional subunit architecture of the regulatory enzyme aspartate transcarbamoylase; two
different views. This allosteric regulatory enzyme has two catalytic clusters, each with three catalytic polypeptide
chains, and three regulatory clusters, each with two regulatory polypeptide chains. The catalytic polypeptides in
each cluster are shown in shades of blue and purple. Binding sites for allosteric modulators are found on the
regulatory subunits (shown in white and red). Modulator binding produces large changes in enzyme conformation
and activity.

Other differences between nonregulated enzymes and allosteric enzymes involve kinetic
properties. Allosteric enzymes show relationships between V0 and [S] that differ from normal
Michaelis-Menten behavior. They do exhibit saturation with the substrate when [S] is sufficiently
high, but for some allosteric enzymes, when V0 is plotted against [S] (Fig 4) a sigmoid saturation

curve results, rather than the hyperbolic curve shown by nonregulatory enzymes. Although we
can find a value of [S] on the sigmoid saturation curve at which V 0 is half maximal, we cannot
refer to it with the designation Km because the enzyme does not follow the hyperbolic MichaelisMenten relationship. Instead the symbol [S]0.5 or K0.5 is often used to represent the substrate
concentration giving half maximal velocity of the reaction catalyzed by an allosteric enzyme (Fig
4).

Figure 4: Substrate-activity curves for representative allosteric enzymes. Three examples of complex responses
given by allosteric enzymes to their modulators. (a) The sigmoid curve given by a homotropic enzyme, in which the
substrate also serves as a positive (stimulatory) modulator. Note that a relatively small increase in [S] in the steep
part of the curve can cause a very large increase in V0. Note also the resemblance to the oxygen-saturation curve of
hemoglobin (see Fl 7-28). (b) The effects of a positive modulator
a negative modulator
, and no
modulator

on an allosteric enzyme in which K0.5 is modulated without a change in Vmax. (c) A less

common type of modulation, in which Vmax is modulated with K0.5 nearly constant.

Sigmoid kinetic behavior generally reflects cooperative interactions between multiple protein
subunits. In other words, changes in the structure of one subunit are translated into structural
changes in adjacent subunits, an effect that is mediated by noncovalent interactions at the
subunit-subunit interface. The principles are similar to those discussed for cooperativity in
oxygen binding to the nonenzyme protein hemoglobin. Homotropic allosteric enzymes generally
have multiple subunits. In many cases the same binding site on each subunit functions as both
the active site and the regulatory site. The substrate can function as a positive modulator (an
activator) because the subunits act cooperatively. The binding of one molecule of the substrate to
one binding site alters the enzyme's conformation and greatly enhances the binding of
subsequent substrate molecules. This accounts for the sigmoid rather than hyperbolic increase in
V0 with increasing [S].

With heterotropic enzymes, in which the modulator is a metabolite other than the substrate itself,
it is difficult to generalize about the shape of the substrate-saturation curve. An activator may
cause the substrate-saturation curve to become more nearly hyperbolic, with a decrease in K 0.5
but no change in Vmax, thus resulting in an increased reaction velocity at a fixed substrate
concentration ( V0) is higher for any value of [S]) (Fig 4b). Other allosteric enzymes respond to
an activator by an increase in Vmax, with little change in K0.5 (Fig 4c). A negative modulator (an
inhibitor) may produce a more sigmoid substrate-saturation curve, with an increase in K 0.5 (Fig 4
b). Allosteric enzymes therefore show different kinds of responses in their substrate-activity
curves because some have inhibitory modulators, some have activating modulators and some
have both.
TWO MODELS EXPLAIN THE KINETIC BEHAVIOR OF ALLOSTERIC ENZYMES
The sigmoidal dependence of V0 on [S] reflects subunit cooperativity, and has inspired two
models to explain these cooperative interactions.
THE SYMMETRY MODEL: proposed by Jacques Monod and colleagues in 1965. An
allosteric enzyme can exist in only two conformations, active and inactive (Fig. 5a). All subunits
are in the active form or all are inactive. Every substrate molecule that binds increases the
probability of a transition from the inactive to the active state.
THE SEQUENTIAL MODEL: (Fig. 5b), proposed by Koshland in 1966. There are still two
conformations, but subunits can undergo the conformational change individually. Binding of
substrate increases the probability of the conformational change. A conformational change in one
subunit makes a similar change in an adjacent subunit, as well as the binding of a second
substrate molecule, more likely. There are more potential intermediate states in this model than
in the symmetry model. The two models are not mutually exclusive; the symmetry model may be
viewed as the "all-or-none" limiting case of the sequential model. The precise mechanism of
allosteric interaction has not been established. Different allosteric enzymes may have different
mechanisms for cooperative interactions.

Figure 5: Two general models for the interconversion of inactive and active forms of allosteric enzymes. Four
subunits are shown because the model was originally proposed for the oxygen-carrying protein hemoglobin. In the
symmetry, or all-ornone, model (a) all the subunits are postulated to be in the same conformation, either all
affinity or inactive) or all

(high affinity or active). Depending on the equilibrium, K l, between

and

(low
forms,

the binding of one or more substrate (S) molecules will pull the equilibrium toward the
form. Subunits with
bound S are shaded. A possible pathway is given by the gray shading. In the sequential model (b) each individual
subunit can be in either the
or
form. A very large number of conformations is thus possible, but the shaded
pathway (diagonal arrows) is the most probable route.

COVALENTLY MODULATED ENZYME:

Here, active and inactive forms of the enzyme are interconverted by covalent modification of
their structures which are catalysed by other enzymes. Modifying groups include phosphate,
adenosine monophosphate, uridine monophosphate, adenosine diphosphate ribose, and methyl
groups. These are generally covalently linked to and removed from the regulatory enzyme by
separate enzymes. Some examples are given below.

Figure 1Some well-studied examples of enzyme modification reactions

An important example of regulation by covalent modification is glycogen phosphorylase (Mr
94,500) of muscle and liver, which catalyzes the reaction
(Glucose)n + Pi

(glucose)n-1 + glucose-1-phosphate

The glucose-1-phosphate so formed can then be broken down into lactate in muscle or converted
to free glucose in the liver. Glycogen phosphorylase occurs in two forms: the active form
phosphorylase a and the relatively inactive form phosphorylase b (Fig.). Phosphorylase a has
two subunits, each with a specific Ser residue that is phosphorylated at its hydroxyl group. These
serine phosphate residues are required for maximal activity of the enzyme. The phosphate
groups can be hydrolytically removed from phosphorylase a by a separate enzyme called
phosphorylase phosphatase:
Phosphorylase a + 2H2O

phosphorylase b + 2Pi

Figure Regulation of glycogen phosphorylase activity by covalent modification. In the active form of the enzyme,
phosphorylase a, specific Ser residues, one on each subunit, are in the phosphorylated state. Phosphorylase a is
converted into phosphorylase b, which is relatively inactive, by enzymatic loss of these phosphate groups, promoted
by phosphorylase phosphatase. Phosphorylase b can be reactivated to form phosphorylase a by the action of
phosphorylase kinase.

In this reaction phosphorylase a is converted into phosphorylase b by the cleavage of two serinephosphate covalent bonds.
Phosphorylase b can in turn be reactivated-covalently transformed back into active
phosphorylase a-by another enzyme, phosphorylase kinase, which catalyzes the transfer of
phosphate groups from ATP to the hydroxyl groups of the specific Ser residues in phosphorylase
b:
2ATP + phosphorylase b

2ADP + phosphorylase a

The breakdown of glycogen in skeletal muscles and the liver is regulated by variations in the
ratio of the two forms of the enzyme. The a and b forms of phosphorylase differ in their
quaternary structure; the active site undergoes changes in structure and, consequently, changes in
catalytic activity as the two forms are interconverted.
Some of the more complex regulatory enzymes are located at particularly crucial points in
metabolism, so that they respond to multiple regulatory metabolites through both allosteric and

covalent modification. Glycogen phosphorylase is an example. Although its primary regulation is

through covalent modification, it is also modulated in a noncovalent, allosteric manner by AMP,
which is an activator of phosphorylase b, and several other molecules that are inhibitors.
Glutamine synthetase of E. coli, one of the most complex regulatory enzymes known, provides
examples of regulation by allostery, reversible covalent modification, and regulating proteins. It
has at least eight allosteric modulators.

ACTIVATION OF AN ENZYME BY PROTEOLYTIC CLEAVAGE

This is a somewhat different type of regulatory mechanism. An inactive precursor of the enzyme,
called a zymogen, is cleaved to form the active enzyme. Many proteolytic enzymes (proteases)
of the stomach and pancreas are regulated this way. Chymotrypsin and trypsin are initially
synthesized as chymotrypsinogen and trypsinogen, respectively (Fig. 8-30).

Figure 8-30 Activation of the zymogens of chymotrypsin and trypsin by proteolytic cleavage. The bars represent
the primary sequence of the polypeptide chains. Amino acids at the termini of the polypeptide fragments generated
by cleavage are indicated below the bars. The numbers represent the positions of the amino acids in the primary
sequence of chymotrypsinogen or trypsinogen. (The amino-terminal amino acid is number 1. )

Specific cleavage causes conformational changes that expose the enzyme active site. Because
this type of activation is irreversible, other mechanisms are needed to inactivate these enzymes.
Proteolytic enzymes are inactivated by inhibitor proteins that bind very tightly to the enzyme
active site. Pancreatic trypsin inhibitor (Mr 6,000) binds to and inhibits trypsin; a~-antiproteinase
(Mr 53,000) primarily inhibits elastase. An insufficiency of al-antiproteinase, believed to be

caused by exposure to cigarette smoke, leads to lung damage and the condition known as
emphysema.
Other examples of zymogen activation occur in hormones, connective tissue, and the bloodclotting system. The hormone insulin is produced by cleavage of proinsulin, collagen is initially
synthesized as a soluble precursor called procollagen, and blood clotting is mediated by a
complicated cascade of zymogen activations.
REFERENCES
1. Branden, C., and J. Tooze. Introduction to Protein Structure. New York: Garland, 1991.
2. Lehninger, A.L., D.L. Nelson, and M.M. Cox. Principles ofBiochemistry. 2nd ed. New
York: Worth, 1993.
3. Kraut, J. "How Do Enzymes Work?" Science 242 (1988): 533-540.

QUESTIONS.
1. Explain in your own words how a feedback inhibitor works.

CHAPTER FIVE
MOLECULAR MODELS FOR ALLOSTERISM

Models of allosteric regulation

A - Active site
B - Allosteric site
C - Substrate
D - Inhibitor
E - Enzyme
This is a diagram of allosteric regulation of an enzyme.
Most allosteric effects can be explained by the concertedMWC model put forth by Monod,
Wyman, and Changeux,[2] or by the sequential model described by Koshland, Nemethy,
and Filmer.[3] Both postulate that enzyme subunits exist in one of two conformations,

tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the
tense state. The two models differ most in their assumptions about subunit interaction and the
preexistence of both states.

Concerted model
The concerted model of allostery, also referred to as the symmetry model or MWC model,
postulates that enzyme subunits are connected in such a way that a conformational change in one
subunit is necessarily conferred to all other subunits. Thus, all subunits must exist in the same
conformation. The model further holds that, in the absence of any ligand (substrate or otherwise),
the equilibrium favors one of the conformational states, T or R. The equilibrium can be shifted to
the R or T state through the binding of one ligand (the allosteric effector or ligand) to a site that
is different from the active site (the allosteric site).

Sequential model
The sequential model of allosteric regulation holds that subunits are not connected in such a way
that a conformational change in one induces a similar change in the others. Thus, all enzyme
subunits do not necessitate the same conformation. Moreover, the sequential model dictates that
molecules of substrate bind via an induced fit protocol. In general, when a subunit randomly
collides with a molecule of substrate, the active site, in essence, forms a glove around its
substrate. While such an induced fit converts a subunit from the tensed state to relaxed state, it
does not propagate the conformational change to adjacent subunits. Instead, substrate-binding at
one subunit only slightly alters the structure of other subunits so that their binding sites are more
receptive to substrate. To summarize:

subunits need not exist in the same conformation

molecules of substrate bind via induced-fit protocol

conformational changes are not propagated to all subunits

Morpheein model
The morpheein model of allosteric regulation is a dissociative concerted model. [4]
A morpheein is a homo-oligomeric structure that can exist as an ensemble of physiologically
significant and functionally different alternate quaternary assemblies. Transitions between
alternate morpheein assemblies involve oligomer dissociation, conformational change in the
dissociated state, and reassembly to a different oligomer. The required oligomer disassembly step
differentiates the morpheein model for allosteric regulation from the classic MWC and KNF
models. Porphobilinogen synthase (PBGS) is the prototype morpheein.

Models of allosteric regulation

The sigmoidal dependence of V0 on [S] reflects subunit co-operativity, has inspired two models
to explain these cooperative interactions. Most allosteric effects can be explained by the
concerted MWC model put forth by Monod, Wyman, and Changeux, or by the sequential model
described by Koshland, Nemethy, and Filmer. Both postulate that enzyme subunits exist in one
of two conformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more
readily than those in the tense state. The two models (figure-9) differ most in their assumptions
about subunit interaction and the pre-existence of both states.
The concerted model
The concerted model of allostery, postulates that enzyme subunits are connected in such a way
that a conformational change in one subunit is necessarily conferred to all other subunits or in
other words, all subunits must be in the same conformation. As mentioned earlier that allosteric
enzymes can exist in two states, i.e., relaxed (R state) and tight (T state). In this two-state model,
all the subunits of an oligomer must be in the same state (they all change together) and is
therefore termed the concerted model . T state predominates in the absence of substrate S and S
bind more tighter to R than T. The T and R state equilibrium depends upon the concentration of
the substrate. At high substrate concentration, more enzymes are found in the R state, whereas at
low substrate concentration, the enzymes are found in the T state. The equilibrium can be shifted
to the R or T state through the binding of the allosteric effector (activator or inhibitor) to the
allosteric site. Activator and inhibitor bind to R and T states respectively (Figure-8). In this
symmetry model, binding of ligand to one subunit always assists the binding of the same ligand
to the next subunit, i.e, only positive co-operativity is possible here. Heterotropic interactions
could either be positive or negative.

Figure 8: The concerted model for allosteric regulation

The sequential model for allosteric regulation

The sequential model (figure-9) of allosteric regulation holds that subunits are not connected in
such a way that a conformational change in one subunit induces a similar change in the other
subunits. Thus, all enzyme subunits do not necessitate the same conformation. Subunits may
undergo individual sequential changes in conformation. Subunits can interact in different
conformations. The sequential model says that molecules of substrate bind via an induced-fit
hypothesis. When a subunit randomly collides with a molecule of substrate, the active site, in
essence, forms a glove around its substrate. While such an induced fit of the substrate and a
subunit, cause a conformation change (Figure-9) in the subunit, converting it from the tensed
state to relaxed state (T form to R form), and increasing the sites available to the substrate. The
alteration of conformation of one subunit by substrate binding is transmitted to other subunits by
subunit intreractions, and symmetry needn't be conserved. Change induced by binding of
substrate to one subunit can increase or decrease substrate binding to other subunits, i.e., the
ligand-induced conformational change in one subunit can affect the adjoining subunit. That
means both positive and negative homotropic interactions are possible in this model.
Heterotropic interactions could either be positive or negative.

Figure 9: The sequential model for allosteric regulation

References
1

Monod, J., Wyman, J, Changeux, J.P. (1965). On the nature of allosteric transitions: a
plausible model. J Mol Biol. 12:88-118.

Gohara, D.W., Di Cera, E. (2011). Allostery in trypsin-like proteases suggests new

therapeutic strategies. "Trends Biotechnol".

Z. Huang, L. Zhu, Y. Cao, G. Wu, X. Liu, et al (2011) ASD: a comprehensive database of

allosteric proteins and modulators. Nucleic Acids Res Volume 39, D663-669

Koshland DE Jr, Nmethy G, Filmer D. (1966). Comparison of experimental binding data

and theoretical models in proteins containing subunits. Biochemistry5(1):365-85.

. Berg, Jeremy H; Tymoczko, John L; Stryer, Lubert. Biochemistry, 6th Edn. W.H.
Freeman Company, New York.

Lehlinger Albert.L, Cox Michael M, Nelson David L. Regulatory Enzymes. Principles of

Biochemistry (225-232), 4th Edn. W. H. freeman & company 2004.

Sabrina Laing, Mandy Unger, Friedrich Koch-Nolte and Friedrich Haag (2011). ADPribosylation of arginine: AminoAcids. 41(2): 257-269.

www. ncbi.nlm.nih.gov

www.wikipedia.org

10 Raymond Chang. Physical Chemistry for the biosciences. University Science Books.
2005
11 Biological Sciences Review Notes. Kaplan, Inc. 2007
12 "The Hill equation revisited: uses and misuses." J N Weiss

CHAPTER SIX
MULTIENZYME COMPLEX/SYSTEM
This is the structural and functional entity that is formed by the association of several different
enzymes which catalyze a sequence of closely related reactions. The aggregate may contain one
or more molecules of the given enzymes. In some cases, the enzyme of a sequence of a reaction
may tightly bind to form such a multi-enzyme complex. Examples include
(1) Pyrvate dehydrogenase complex
(2) Pyruvate carboxylase
(3) Fatty acid synthase
Pyruvate dehydrogenase complex (P.D.C)
This enzyme catalyze the oxidative decarboxylation of pyruvate to acetylCoA. It is an organized
assembly of 3 types of enzyme. The reaction catalyzed is summarized thus,
Pyruvate + CoA Acetyl CoA +CO+ NADH
It has cofactors namely, CoA, NAD, thiamine PPO and FAD.
Structural assembly of E-coli PDC studies have these properties:
ENZYME
PYRUVATE DH
COMPONENT
DIHYDROLIPOYL
TRANSACETYLAS
E
DIHYDROLIPOYL
TRANSACETYLAS
E

ABBREVIATION NO OF
CHAIN
S
A or E1
24

PROSTHETI
C
GROUP
TPP

B or E2

12

LIPOAMIDE

C or E3

12

FAD

REACTION
CATALYSED
DECARBOXYLATION
OF PYRUVATE
OXIDATION OF
CARBON 2 UNITS
REGENERATION OF
THE OXIDIZED
FORM
OF
LIPOAMIDE

MECHANISM
COMPLEX

OF

ACTION

OF

PYRUVATE

DEHYDROGENASE

There are 4 steps in the conversion of pyruvate to acetyl CoA .

1 Decarboxylation of pyruvate after its combination with TPP in a reaction catalyzed by E1
(pyruvate dehydrogenase complex)
2 The hydroxyl ethyl ether group attached to TPP is oxidized to form an acetyl group
concomitantly transferred to lipoamide.
(3) The acetyl group is transferred from acetyl lipoamide to CoA to form acetyl CoA.
(4) The oxidized form of lipoamide is regenerated to complete the reaction. NAD is the
oxidant in this reaction catalyzed by E3.

CONTROL OF PYRUVATE DEHYDROGENASE COMPLEX

(1)CONTROL OF PRODUCT INHIBITION: Acetyl CoA and NADH inhibit the enzyme
complex and this is reversible by AMP.
(2) FEED BACK REGULATION BY NUCLEOTIDES: The enzyme is inhibited by GTP and
AMP.
(3) REGULATION BY COVALENT MODIFICATION: The enzyme complex becomes
enzymatically inactive when a specific serine residue of pyruvate dehydrogenase complex is
phosphorylated by ATP.
MECHANISM OF ACTION: PYRUVATE CARBOXYLASE (i.e CATALYTIC
ACTIVITY).
Pyruvate carboxylase catalyses the conversion of pyruvate to oxaloacetate. It has both catalytic
and allosteric properties. It contain a prosthetic group (biotin) which serves as a carrier of
activated co.This biotin is linked to the epsilon amino group of specific lysine residue by an
amide bond. The carboxylation processes occur in two (2) stages thus:
(1) Biotin-energy + ATP + HCOCO-biotin-enz + ADP + pi
(2) Co-biotin-enz + pyruvate-biotin-enz + oxaloacetate
The carboxyl group in the carboxy-biotin enzyme intermediate is bounded to N-1 of biotin ring.
The carboxyl group Is activated thus:
Co-biotin-enz + HCO+ biotin-enz
The activated carboxyl group is then tranfered from the carboxylbiotin to pyruvate to form
oxaloacetate. The long flexible chain enables this prosthetic group to rotate from one active site
of the enzyme (the ATP-biocarbonate site) to other (the pyruvate site).
CONTROL OF PYRUVATE CARBOXYLASE
The activity of pyruvate carboxylase is dependent largely on the presence of acetyl CoA because
biotin will not be carboxylated unless acetyl coA ( or a closely related acyl CoA) is bound to the
enzyme. A high level of acetyl CoA signals the need for more oxaloacetate. If there is surplus of
ATP , oxaloacetate will be cosumed in gluconeogenesis. If there is deficiency of ATP,
oxaloacetate will enter into TCA cycle upon condensing with acetyl CoA to form citrate.

CHAPTER SEVEN
ENZYME ASSAY TECHNIQUES
Assay simply means measurement of the enzymatic activity that is based on the determination of
either the rate of formation of the product or the rate of utilization or disappearance of reactant or
substrate under control conditions. Most assays are carried out at 30C-37C. Adequate buffering
capacity is always being ensured. Apparatus used must be clean. Analytical assays may be
classified as (1) continous (2) discontinuous.
In order to standardize the report on enzyme activities, the commission of enzyme of the
international union of biochemistry defined a standard unit i.e International unit (I.U) of enzyme
as the amount of enzyme that catalysed the formation of 1 micro mole of product per minute
under defined condition. The concentration of enzyme in an impure preparation is expressed in
terms of units/ml while the specificity activity is expressed as units/mg protein.
In most cases, as the enzyme is being purified, its specific activity is expected to increase to
maximum.
Kcat: is defined as the amount of enzyme that catalyze I mole of substrate per second.
Turnover number : is defined as the number of moles of substrate transformed per minute per
second.
There exist different enzyme assay techniques, however, irrespective of the principle of the
chosen method, the enzyme assay requires the use of excess substrate (zero order kinetics at least
equal to 10Km) and an appropriate control is required. This control is in all respect the same as
the test assay but lacks either the enzyme or substrate. Both the test and the control must be
subjected to the same experimental condition.

TYPES OF ENZYME ASSAY TECHNIQUES

(1) VISIBLE AND UV SPECTROPHOTOMETRIC METHOD
(2) SPECTROFLUORIMETRY
(3) RADIOISOTOPE
(4) IMMUNOCHEMICAL METHOD
(5) MACRO CALORIMETRIC METHOD
(6) MANOMETRIC METHOD

(7) COUPLE ASSAY METHOD/TECHNIQUES

ENZYME ASSAYS
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital
for the study of enzyme kinetics and enzyme inhibition.

Enzyme units
Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or
measured in terms of activity, in enzyme units.

Enzyme activity
Enzyme activity = moles of substrate converted per unit time = rate reaction volume. Enzyme
activity is a measure of the quantity of active enzyme present and is thus dependent on
conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s1, but this is an
excessively large unit. A more practical and commonly used value is 1 enzyme unit (U) = 1
mol min1. 1 U corresponds to 16.67 nanokatals.
Enzyme activity as given in katal generally refers to that of the assumed natural target substrate
of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such
as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in
milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the
enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.

Specific activity
The specific activity of an enzyme is another common unit. This is the activity of an enzyme per
milligram of total protein (expressed in mol min 1mg1). Specific activity gives a measurement
of the activity of the enzyme. It is the amount of product formed by an enzyme in a given
amount of time under given conditions per milligram of total protein. Specific activity is equal to
the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The
SI unit is katal kg1, but a more practical unit is mol mg 1 min1. Specific activity is a measure
of enzyme processivity, at a specific (usually saturating)substrate concentration, and is usually
constant for a pure enzyme. For elimination of errors arising from differences in cultivation
batches and/or misfolded enzyme etc. an active site titration needs to be done. This is a measure
of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by
employing an irreversible inhibitor. The specific activity should then be expressed as mol min 1
mg1 active enzyme.

Related terminology

The rate of a reaction is the concentration of substrate disappearing (or product produced) per
unit time (molL 1s 1).
The % purity is 100% (specific activity of enzyme sample / specific activity of pure enzyme).
The impure sample has lower specific activity because some of the mass is not actually enzyme.
If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower
specific activity, allowing purity to be calculated.

Types of assay
All enzyme assays measure either the consumption of substrate or production of product over
time. A large number of different methods of measuring the concentrations of substrates and
products exist and many enzymes can be assayed in several different ways. Biochemists usually
study enzyme-catalysed reactions using four types of experiments:
Initial rate experiments. When an enzyme is mixed with a large excess of the substrate,
the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction
achieves a steady-state kinetics in which enzyme substrate intermediates remains
approximately constant over time and the reaction rate changes relatively slowly. Rates
are measured for a short period after the attainment of the quasi-steady state, typically by
monitoring the accumulation of product with time. Because the measurements are carried
out for a very short period and because of the large excess of substrate, the approximation
free substrate is approximately equal to the initial substrate can be made. The initial rate
experiment is the simplest to perform and analyze, being relatively free from
complications such as back-reaction and enzyme degradation. It is therefore by far the
most commonly used type of experiment in enzyme kinetics.

Progress curve experiments. In these experiments, the kinetic parameters are

determined from expressions for the species concentrations as a function of time. The
concentration of the substrate or product is recorded in time after the initial fast transient
and for a sufficiently long period to allow the reaction to approach equilibrium. We note
in passing that, while they are less common now, progress curve experiments were
widely used in the early period of enzyme kinetics.

Transient kinetics experiments. In these experiments, reaction behaviour is tracked

during the initial fast transient as the intermediate reaches the steady-state kinetics period.
These experiments are more difficult to perform than either of the above two classes
because they require specialist techniques (such as flash photolysis of caged
compounds) or rapid mixing (such as stopped-flow, quenched flow or continuous
flow).

Relaxation experiments. In these experiments, an equilibrium mixture of enzyme,

substrate and product is perturbed, for instance by a temperature, pressure or pH
jump, and the return to equilibrium is monitored. The analysis of these experiments
requires consideration of the fully reversible reaction. Moreover, relaxation experiments
are relatively insensitive to mechanistic details and are thus not typically used for
mechanism identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous
assays, where the assay gives a continuous reading of activity, and discontinuous assays, where
samples are taken, the reaction stopped and then the concentration of substrates/products
determined.

Temperature-controlled cuvette holder in a spectrophotometer.

Continuous assays
Continuous assays are most convenient, with one assay giving the rate of reaction with no further
work necessary. There are many different types of continuous assays.

Spectrophotometric
In spectrophotometric assays, you follow the course of the reaction by measuring a change
in how much light the assay solution absorbs. If this light is in the visible region you can actually
see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a
redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.
UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in
their reduced forms, but do not in their oxidized forms. An oxidoreductase using NADH
as a substrate could therefore be assayed by following the decrease in UV absorbance at a
wavelength of 340 nm as it consumes the coenzyme.

Direct versus coupled assays

Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.

Even when the enzyme reaction does not result in a change in the absorbance of light, it can still
be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here,
the product of one reaction is used as the substrate of another, easily detectable reaction. For
example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed
by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6phosphate dehydrogenase.

Fluorometric
Fluorescence is when a molecule emits light of one wavelength after absorbing light of a
different wavelength. Fluorometric assays use a difference in the fluorescence of substrate
from product to measure the enzyme reaction. These assays are in general much more sensitive
than spectrophotometric assays, but can suffer from interference caused by impurities and the
instability of many fluorescent compounds when exposed to light.
An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH.
Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation
reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an
increase. Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are
also available, such as 4-methylumbelliferyl--D-galactoside for assaying -galactosidase.

Calorimetric
Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These
assays are very general, since many reactions involve some change in heat and with use of a
microcalorimeter, not much enzyme or substrate is required. These assays can be used to
measure reactions that are impossible to assay in any other way.

Chemiluminescent
Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions
produce light and this can be measured to detect product formation. These types of assay can be
extremely sensitive, since the light produced can be captured by photographic film over days or
weeks, but can be hard to quantify, because not all the light released by a reaction will be
detected.
The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a
common method of detecting antibodies in western blotting. Another example is the enzyme
luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.

Chemiluminescence of Luminol

Light Scattering
Static light scattering measures the product of weight-averaged molar mass and
concentration of macromolecules in solution. Given a fixed total concentration of one or more
species over the measurement time, the scattering signal is a direct measure of the weightaveraged molar mass of the solution, which will vary as complexes form or dissociate. Hence the
measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering
assays of protein kinetics is a very general technique that does not require an enzyme.

Microscale Thermophoresis
Microscale Thermophoresis (MST) measures the size, charge and hydration entropy of
molecules/substrates in real time. The thermophoretic movement of a fluorescently labeled
substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be
measured with high time resolution in real time. The material consumption of the all optical
MST method is very low, only 5 l sample volume and 10nM enzyme concentration are needed
to measure the enzymatic rate constants for activity and inhibition. MST allows to measure the
modification of two different substrates at once (multiplexing) if both substrates are labeled
with different fluorophores. Thus substrate competition experiments can be performed.

Discontinuous assays
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the
amount of product production or substrate consumption is measured in these samples.

Radiometric
Radiometric assays measure the incorporation of radioactivity into substrates or its release
from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P,
35
S and 125I. Since radioactive isotopes can allow the specific labelling of a single atom of a

substrate, these assays are both extremely sensitive and specific. They are frequently used in
biochemistry and are often the only way of measuring a specific reaction in crude extracts (the
complex mixtures of enzymes produced when you lyse cells).
Radioactivity is usually measured in these procedures using a scintillation counter.

Chromatographic
Chromatographic assays measure product formation by separating the reaction mixture into its
components by chromatography. This is usually done by high-performance liquid
chromatography (HPLC), but can also use the simpler technique of thin layer
chromatography. Although this approach can need a lot of material, its sensitivity can be
increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay
sensitivity has also been increased by switching protocols to improved chromatographic
instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a fewfold
higher
than
HPLC
instruments
(see
High-performance
liquid
chromatography#Pump_pressure).

Factors to control in assays

Salt Concentration: Most enzymes cannot tolerate extremely high salt concentrations.
The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active
in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic
(salt loving) algae and bacteria.

Effects of Temperature: All enzymes work within a range of temperature specific to the
organism. Increases in temperature generally lead to increases in reaction rates. There is a
limit to the increase because higher temperatures lead to a sharp decrease in reaction
rates. This is due to the denaturating (alteration) of protein structure resulting from the
breakdown of the weak ionic and hydrogen bonding that stabilize the three
dimensional structure of the enzyme active site.[11] The "optimum" temperature for
human enzymes is usually between 35 and 40 C. The average temperature for humans is
37 C. Human enzymes start to denature quickly at temperatures above 40 C. Enzymes
from thermophilicarchaea found in the hot springs are stable up to 100 C.[12]
However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate
observed at any temperature is the product of two rates, the reaction rate and the
denaturation rate. If you were to use an assay measuring activity for one second, it would
give high activity at high temperatures, however if you were to use an assay measuring
product formation over an hour, it would give you low activity at these temperatures.

Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All
have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the
three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds.
Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works
best at a pH of 2 and trypsin at a pH of 8.

Substrate Saturation: Increasing the substrate concentration increases the rate of

reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme
is saturated when the active sites of all the molecules are occupied most of the time. At
the saturation point, the reaction will not speed up, no matter how much additional
substrate is added. The graph of the reaction rate will plateau.

Level of crowding, large amounts of macromolecules in a solution will alter the

rates and equilibrium constants of enzyme reactions, through an effect called
macromolecular crowding.

REFERENCES

QUESTIONS
1. (a) Define Enzyme Assay
(b) Explain the Continuous and Discontinuous methods of Enzyme Assay.
2. Write a short note on the Factors to consider when carrying out an enzyme
assay.
3. Differentiate between Direct and coupled assays.
4. What are the Instruments/methods for measuring enzyme activities?

REFERENCES
CONTACT: Keith, W and John.W (1995) Practical Biochemistry: Principle and
technique. 4th edition; Cambridge University Press, pp 209-216.

CHAPTER EIGHT

CRITERIA FOR DETERMINING PURITY OF ENZYMES

CHAPTER NINE

ENZYME RECONSTITUTION.
Reconstitution:
Your enzyme may have been damaged in some way, and is inactive. You can try to
reconstitute it, i.e., try to give it back its structure and activity. You could, for
instance, try to denature it completely in a 8 M urea solution, and then dialyse the
urea out to renature it.
Or it could be oxidized, and therefore be inactive. Then you could try to reduce it
with an SH reagent.

Abstract

Send to:
Curr Opin Pediatr. 2007 Dec;19(6):628-35.

Enzyme reconstitution/replacement therapy

for lysosomal storage diseases.
Burrow TA1, Hopkin RJ, Leslie ND, Tinkle BT, Grabowski GA.

Author information
Abstract
PURPOSE OF REVIEW:
Over the past 15 years, the lysosomal storage diseases have become paradigms for the specific
treatment of monogenic disorders, particularly those affecting children. This review summarizes
the phenotypes and recent literature regarding enzyme reconstitution (replacement) therapy and
outcomes for such treatable lysosomal storage diseases: Gaucher disease, Fabry disease, Pompe
disease and the mucopolysaccharidoses.
RECENT FINDINGS:
Recent clinical trials have shown that enzyme reconstitution therapy effectively treats many of
the manifestations of the lysosomal storage diseases. When initiated early in the disease course,
enzyme reconstitution therapy can reverse some disease manifestations, but may not completely
alleviate the disease progression. Enzyme reconstitution therapy is generally well tolerated.
Many adverse events are antibody-related, but can be managed without requiring cessation of
enzyme reconstitution therapy. Documented IgE reactions, i.e. anaphylactoid, are quite rare
(fewer than 1%).
SUMMARY:

Enzyme reconstitution therapy is a safe and effective treatment modality available for several of
the lysosomal storage diseases. Owing to the short history of enzyme reconstitution therapy, the
long-term outcomes of enzyme reconstitution therapy-treated individuals are unknown and
require further investigation. Medical professionals must learn to identify patients likely to
benefit from these life-changing therapies so as to prevent many of the devastating, irreversible
complications of the lysosomal storage diseases.

CHAPTER TEN
REGULATION OF ENZYME ACTIVITY
In every metabolic pathway, the activity of at least one enzyme is subject to regulation so that the
flux of material through the pathway can be controlled. There are at least 4 means of control
namely:
(1) Allosteric control
(2) Feedback inhibition
(3) Control by reversible covalent modification
(4) Control by irreversible covalent modification
ALLOSTERIC CONTROL: Some enzymes are reversibly inhibited or activated by the
presence of metabolites that are not their substrate or product. These metabolites, if inhibitory are
normally distant products of the pathway, thereby providing negative feedback for the activity of
the pathway. The enzymes controlled in this way usually have additional binding site other than
the active or substrate binding site. The binding of inhibitor or activators at distant site from
active site often brought about conformational changes in the enzyme molecule which may
decrease or increase its catalytic activity. Thus, allosteric enzymes usually compose of subunits
and use to have multiple interacting active centres and often shows sigmoidal graph of initial rate
versus [S] and therefore do not obey Michaelis Menten kinetics.
FEEDBACK INHIBITION: This is a means by which biosynthetic pathways are regulated and
involves the process whereby and products or near end products control the metabolic flux by
inhibiting one or more of the enzyme at the early part of the pathway. Often maximum feedback
inhibition is attained only by the combined actions of multiple end products. Feedback inhibition
could be (1) sequential (2) concerted (3)cumulative (4) co-operative

ILLUSTRATION
CONTROL BY REVERSIBLE COVALENT MODIFICATION: This regulation is often the
response to a signal coming from outside the cells such as response to hormone. In this case, the
enzyme is itself the substrate of other enzymes. One of these modifies the enzyme making it
active while the other reverses the modification thereby is inactive. A typical example is the
control of glycogen phosphorylase through phosphorylation and dephosphorylation of specific
serine residue. The enzyme exist in two forms i.e glycogen phosphorylase a (active) and
glycogen phosphorylase b (inactive).
CONTROL BY IRREVERSIBLE COVALENT MODIFICATION
The best known example of the enzyme that exhibit /undergo this mechanism or modification is
a protease enzyme named chymotrypsinogen. The enzyme, when reacted with diisopropyl
phosphofluoridate becomes inactivated because of the reaction of serine-195 with chemical
coupled with the formation of covalent linkage and elimination of HF. Also, activation of
zymogen is another example of this type of modification. For instance, activation of trypsinogen
by enteropeptidase will result in formation of trypsin, which also acts on chymotrypsinogen and
converts its to chymotrypsin

PRODUCTION, ISOLATION, PURIFICATION AND

CHARACTERISATION OF ENZYMES
INTRODUCTION

The sources of proteins are plant, animal, or microbial cells. Certain tissues
such as pancreas glands or liver are particularly rich in enzymes. However,
some of the enzymes in the mixture when cells are disrupted are proteases.
They digest proteins. Enzymes are proteins, and proteases can digest
themselves. We can take advantage of the change in enzyme activity with pH by
changing to a pH where the protease activity is low. The disruption of cells to
release enzymes can be carried out at low pH and cold temperatures to
minimize losses due to proteolytic activity.
Proteins have great specificity that can be used to attach them to other
molecules. Attaching an protein to a solid by exploiting this specificity is called
affinity chromatography. Sometimes quite pure enzymes can be recovered from
a mixture by affinity chromatography, but more often there is a preliminary
separation. Each binding site can attach to only one enzyme molecule, so it can
be expensive to provide enough of the binding agent.
A very common preliminary separation comes from adding ammonium sulfate
in stages. Different proteins precipitate at different salt concentrations, and
this divides the enzymes and other proteins into fractions. There are other salts
for precipitation, and polyethylene glycol is an effective organic precipitant.
The fraction with the desired enzyme also contains much salt. Enzymes form
colloidal suspensions rather than true solutions, but we make the common
mistake of saying that the enzyme is "redissolved" in water. Dialysis with

membranes through which the enzyme cannot pass is the popular method for
removing the salt.
Chromatographic separation is covered in much detail in other pages and will
not be discussed here. New techniques such as displacement chromatography
can actually concentrate the enzyme, but conventional chromatography that
flushes it through the column causes much dilution. After concentration by
means such as forcing the water out through a finely pored membrane, freeze
drying is a popular method for producing a stable product.

OBTAIN CELLS BY FERMENTATION OR PURCHASE OF PLANT OR ANIMAL

ORGAN

RELEASE PROTEINS FROM CELLS. RUPTURE BY SHEAR USUALLY.

REMOVE CELL DEBRIS BY FILTRATION OR CENTRIFUGATION

PRECIPITATE PROTEINS WITH AMMONIUM SULPHATE OR POLYETHYLENE

GLYCOL

COLLECT PRECIPITATE AND RESUSPEND FOR SALT REMOVAL BY DIALYSIS

ADVANCE TO CHROMATOGRAPHIC PURIFICATION, USUALLY BY GEL

IF DILUTED BY CHROMATOGRAPY, POOL IS MEMBRANE CONCENTRATED

FREEZE DRYING IS COMMON METHOD TO GET FINAL PRODUCT

METHODS OF PROTEIN PURIFICATION

The methods used in protein purification can roughly be divided into analytical
and preparative methods. The distinction is not exact, but the deciding factor is
the amount of protein that can practically be purified with that method.
Analytical methods aim to detect and identify a protein in a mixture, whereas
preparative methods aim to produce large quantities of the protein for other
purposes, such as structural biology or industrial use. In general, the
preparative methods can be used in analytical applications, but not the other
way around.

EXTRACTION
Depending on the source, the protein has to be brought into solution by
breaking the tissue or cells containing it. There are several methods to achieve
this: Repeated freezing and thawing, sonication, homogenization by high
pressure, filtration , or permeabilization by organic solvents. The method of
choice depends on how fragile the protein is and how sturdy the cells are. After
this extraction process soluble proteins will be in the solvent, and can be
separated from cell membranes, DNA etc. by centrifugation. The extraction
process also extracts proteases, which will start digesting the proteins in the
solution. If the protein is sensitive to proteolysis, it is usually desirable to
proceed quickly, and keep the extract cooled, to slow down proteolysis.

PRECIPITATION AND DIFFERENTIAL SOLUBILIZATION

In bulk protein purification, a common first step to isolate proteins is
precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding
increasing amounts of ammonium sulfate and collecting the different fractions
of precipitate protein. Ammonium sulphate can be removed by dialysis.The
hydrophobic groups on the proteins gets exposed to the atmosphere and it

attracts other protein hydrophobic groups and gets aggregated. Protein

precipitated will be large enough to be visible. One advantage of this method is
that it can be performed inexpensively with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of
integral membrane proteins requires disruption of the cell membrane in order
to isolate any one particular protein from others that are in the same
membrane compartment. Sometimes a particular membrane fraction can be
isolated first, such as isolating mitochondria from cells before purifying a
protein located in a mitochondrial membrane. A detergent such as sodium
dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep
membrane proteins in solution during purification; however, because SDS
causes denaturation, milder detergents such as Triton X-100 or CHAPS can be
used to retain the protein's native conformation during complete purification.

ULTRACENTRIFUGATION

Centrifugation is a process that uses centrifugal force to separate mixtures of

particles of varying masses or densities suspended in a liquid. When a vessel
(typically a tube or bottle) containing a mixture of proteins or other particulate
matter, such as bacterial cells, is rotated at high speeds, the angular
momentum yields an outward force to each particle that is proportional to its
mass. The tendency of a given particle to move through the liquid because of
this force is offset by the resistance the liquid exerts on the particle. The net
effect of "spinning" the sample in a centrifuge is that massive, small, and dense
particles move outward faster than less massive particles or particles with
more "drag" in the liquid. When suspensions of particles are "spun" in a
centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for
the most massive particles with low drag in the liquid. Non-compacted particles
still remaining mostly in the liquid are called the "supernatant" and can be
removed from the vessel to separate the supernatant from the pellet. The rate
of centrifugation is specified by the angular acceleration applied to the sample,
typically measured in comparison to the g. If samples are centrifuged long
enough, the particles in the vessel will reach equilibrium wherein the particles
accumulate specifically at a point in the vessel where their buoyant density is
balanced with centrifugal force. Such an "equilibrium" centrifugation can allow
extensive purification of a given particle.
Sucrose gradient centrifugation a linear concentration gradient of sugar
(typically sucrose, glycerol, or a silica based density gradient media, like
Percoll) is generated in a tube such that the highest concentration is on the

bottom and lowest on top. Percoll is a trademark owned by GE Healthcare

companies. A protein sample is then layered on top of the gradient and spun at
high speeds in an ultracentrifuge. This causes heavy macromolecules to
migrate towards the bottom of the tube faster than lighter material. During
centrifugation in the absence of sucrose, as particles move farther and farther
from the center of rotation, they experience more and more centrifugal force
(the further they move, the faster they move). The problem with this is that the
useful separation range of within the vessel is restricted to a small observable
window. Spinning a sample twice as long doesn't mean the particle of interest
will go twice as far, in fact, it will go significantly further. However, when the
proteins are moving through a sucrose gradient, they encounter liquid of
increasing density and viscosity. A properly designed sucrose gradient will
counteract the increasing centrifugal force so the particles move in close
proportion to the time they have been in the centrifugal field. Samples
separated by these gradients are referred to as "rate zonal" centrifugations.
After separating the protein/particles, the gradient is then fractionated and
collected.

CHROMATOGRAPHIC METHODS

Chromatographic equipment. Here set up for a size exclusion chromatography.

The buffer is pumped through the column (right) by a computer controlled
device.
Usually a protein purification protocol contains one or more chromatographic
steps. The basic procedure in chromatography is to flow the solution containing
the protein through a column packed with various materials. Different proteins
interact differently with the column material, and can thus be separated by the
time required to pass the column, or the conditions required to elute the
protein from the column. Usually proteins are detected as they are coming off
the column by their absorbance at 280 nm. Many different chromatographic
methods exist:

SIZE EXCLUSION CHROMATOGRAPHY

Chromatography can be used to separate protein in solution or denaturing

conditions by using porous gels. This technique is known as size exclusion
chromatography. The principle is that smaller molecules have to traverse a
larger volume in a porous matrix. Consequentially, proteins of a certain range
in size will require a variable volume of eluent (solvent) before being collected at
the other end of the column of gel.
In the context of protein purification, the eluant is usually pooled in different
test tubes. All test tubes containing no measurable trace of the protein to
purify are discarded. The remaining solution is thus made of the protein to
purify and any other similarly-sized proteins.

SEPARATION BASED ON CHARGE OR HYDROPHOBICITY

Hydrophobic Interaction Chromatography
Resin used in the column are amphiphiles with both hydrophobic and
hydrophilic regions. The hydrophobic part of the resin attracts hydrophobic
region on the proteins. The greater the hydrophobic region on the protein the
stronger the attraction between the gel and that particular protein.

Ion exchange chromatography

Ion exchange chromatography separates compounds according to the nature
and degree of their ionic charge. The column to be used is selected according to
its type and strength of charge. Anion exchange resins have a positive charge
and are used to retain and separate negatively charged compounds, while
cation exchange resins have a negative charge and are used to separate
positively charged molecules.
Before the separation begins a buffer is pumped through the column to
equilibrate the opposing charged ions. Upon injection of the sample, solute
molecules will exchange with the buffer ions as each competes for the binding
sites on the resin. The length of retention for each solute depends upon the
strength of its charge. The most weakly charged compounds will elute first,
followed by those with successively stronger charges. Because of the nature of
the separating mechanism, pH, buffer type, buffer concentration, and
temperature all play important roles in controlling the separation.
Ion exchange chromatography is a very powerful tool for use in protein
purification and is frequently used in both analytical and preparative
separations.

Affinity chromatography

Affinity Chromatography is a separation technique based upon molecular

conformation, which frequently utilizes application specific resins. These resins
have ligands attached to their surfaces which are specific for the compounds to
be separated. Most frequently, these ligands function in a fashion similar to
that of antibody-antigen interactions. This "lock and key" fit between the ligand
and its target compound makes it highly specific, frequently generating a single
peak, while all else in the sample is unretained.
Many membrane proteins are glycoproteins and can be purified by lectin
affinity chromatography. Detergent-solubilized proteins can be allowed to bind

to a chromatography resin that has been modified to have a covalently attached

lectin. Proteins that do not bind to the lectin are washed away and then
specifically bound glycoproteins can be eluted by adding a high concentration
of a sugar that competes with the bound glycoproteins at the lectin binding
site. Some lectins have high affinity binding to oligosaccharides of glycoproteins
that is hard to compete with sugars, and bound glycoproteins need to be
released by denaturing the lectin.

Nickel-affinity column. The resin is blue since it has bound nickel.

Metal binding

A common technique involves engineering a sequence of 6 to 8 histidines into

the N- or C-terminal of the protein. The polyhistidine binds strongly to divalent
metal ions such as nickel and cobalt. The protein can be passed through a
column containing immobilized nickel ions, which binds the polyhistidine tag.
All untagged proteins pass through the column. The protein can be eluted with
imidazole, which competes with the polyhistidine tag for binding to the column,
or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag
for the resin. While this procedure is generally used for the purification of
recombinant proteins with an engineered affinity tag (such as a 6xHis tag or
Clontech's HAT tag), it can also be used for natural proteins with an inherent
affinity for divalent cations.

Immunoaffinity chromatography

A HPLC. From left to right: A pumping device generating a gradient of two

different solvents, a steel enforced column and an apparatus for measuring the
absorbance.
Immunoaffinity chromatography uses the specific binding of an antibody to the
target protein to selectively purify the protein. The procedure involves
immobilizing an antibody to a column material, which then selectively binds
the protein, while everything else flows through. The protein can be eluted by
changing the pH or the salinity. Because this method does not involve
engineering in a tag, it can be used for proteins from natural sources.

Purification of a tagged protein

Adding a tag to the protein such as RuBPS gives the protein a binding affinity it
would not otherwise have. Usually the recombinant protein is the only protein
in the mixture with this affinity, which aids in separation. The most common
tag is the Histidine-tag (His-tag), that has affinity towards nickel or cobalt ions.
Thus by immobilizing nickel or cobalt ions on a resin, an affinity support that
specifically binds to histidine-tagged proteins can be created. Since the protein
is the only component with a His-tag, all other proteins will pass through the
column, and leave the His-tagged protein bound to the resin. The protein is
released from the column in a process called elution, which in this case
involves adding imidazole, to compete with the His-tags for nickel binding, as it
has a ring structure similar to histidine. The protein of interest is now the
major protein component in the eluted mixture, and can easily be separated
from any minor unwanted contaminants by a second step of purification, such
as size exclusion chromatography or RP-HPLC.
Another way to tag proteins is to engineer an antigen peptide tag onto the
protein, and then purify the protein on a column or by incubating with a loose
resin that is coated with an immobilized antibody. This particular procedure is
known as immunoprecipitation. Immunoprecipitation is quite capable of
generating an extremely specific interaction which usually results in binding
only the desired protein. The purified tagged proteins can then easily be
separated from the other proteins in solution and later eluted back into clean
solution.

When the tags are not needed anymore, they can be cleaved off by a protease.
This often involves engineering a protease cleavage site between the tag and the
protein.

HPLC

High performance liquid chromatography or high pressure liquid

chromatography is a form of chromatography applying high pressure to drive
the solutes through the column faster. This means that the diffusion is limited
and the resolution is improved. The most common form is "reversed phase"
hplc, where the column material is hydrophobic. The proteins are eluted by a
gradient of increasing amounts of an organic solvent, such as acetonitrile. The
proteins elute according to their hydrophobicity. After purification by HPLC the
protein is in a solution that only contains volatile compounds, and can easily
be lyophilized.[3] HPLC purification frequently results in denaturation of the
purified proteins and is thus not applicable to proteins that do not
spontaneously refold.

Concentration of the purified protein

A selectively permeable membrane can be mounted in a centrifuge tube. The

buffer is forced through the membrane by centrifugation, leaving the protein in
the upper chamber.

At the end of a protein purification, the protein often has to be concentrated.

Different methods exist.

Lyophilization
If the solution doesn't contain any other soluble component than the protein in
question the protein can be lyophilized (dried). This is commonly done after an
HPLC run. This simply removes all volatile component leaving the proteins
behind.

Ultrafiltration
Ultrafiltration concentrates a protein solution using selective permeable
membranes. The function of the membrane is to let the water and small
molecules pass through while retaining the protein. The solution is forced
against the membrane by mechanical pump or gas pressure or centrifugation.

Analytical
Denaturing-Condition Electrophoresis
Gel electrophoresis is a common laboratory technique that can be used both as
preparative and analytical method. The principle of electrophoresis relies on
the movement of a charged ion in an electric field. In practice, the proteins are
denatured in a solution containing a detergent (SDS). In these conditions, the
proteins are unfolded and coated with negatively charged detergent molecules.
The proteins in SDS-PAGE are separated on the sole basis of their size.
In analytical methods, the protein migrate as bands based on size. Each band
can be detected using stains such as Coomassie blue dye or silver stain.
Preparative methods to purify large amounts of protein, require the extraction
of the protein from the electrophoretic gel. This extraction may involve excision
of the gel containing a band, or eluting the band directly off the gel as it runs
off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis
provides an improved resolution over size exclusion chromatography, but does
not scale to large quantity of proteins in a sample as well as the late
chromatography columns.

Non-Denaturing-Condition Electrophoresis

An important non-denaturing electrophoretic procedure for isolating bioactive

metalloproteins in complex protein mixtures is termed 'quantitative native
continuous polyacrylamide gel electrophoresis (QPNC-PAGE).
References
1.
^
"The
Nobel
Prize
in
Chemistry
1946".
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1946/.
Retrieved 2011-09-19.
2. ^ Ehle H, Horn A (1990). "Immunoaffinity chromatography of enzymes".
Bioseparation 1 (2): 97110. PMID 1368167.
3. ^ Regnier FE (October 1983). "High-performance liquid chromatography
of biopolymers". Science 222 (4621): 24552. doi:10.1126/science.6353575.
PMID 6353575.
http://www.sciencemag.org/cgi/pmidlookup?
view=long&pmid=6353575

QUESTIONS
1. Enumerate the steps involved in purifying enzymes to homogeneity.
2. Explain the steps involved in purifying enzymes to homogeneity.
3. Write short notes on 5 chromatographic methods that can be used in
separating enzymes.
4. How do you confirm whether an enzyme is pure or not?