Enzymes

Objectives
List

general characteristics and classes of enzymes

To understand the following terms; active site, proenzyme,

haloenzyme, apoenzyme, cofactor, prothetic group and
enzyme activity.
To understand the Michaelis-Menton Kinetic theory of
enzyme action
To understand enzyme inhibition and its relevance to
medicine

General Characteristics
Most enzymes are globular protein catalysts that increase the
velocity of a chemical reaction, and are not consumed during
the reaction they catalyze
Non protein enzymes exists RNA molecules (ribozymes)
Linear chains of amino acids folded into 3-D macromolecules
Unique amino acid sequence produces specific enzyme with
unique properties for binding to substrate.
A)Active sites; A special pocket containing amino acid side
chains that create a three-dimensional surface complementary
to the substrate

B) Catalytic efficiency; Most enzyme-catalyzed reactions

are highly efficient. Capable of transforming 100 to 1000
substrate molecules into product each second
C) Specificity; Enzymes are highly specific in the reactions
they catalyse.
D) Regulation; enzymes can be activated or inhibited, so that
the rate of product formation responds to the needs of the cell
E) Location within the cell; Many enzymes are localized in
specific organelles within the cell.

F) Cofactors; Some enzymes require additional chemical

groups for activity. These can be inorganic ions such as Fe2,
Mg2, Mn2, or Zn2 or organic called coenzymes.
Coenzymes are often derivatives of vitamins (e.g. the
coenzyme NAD+ contains niacin).
Haloenzyme is a catalytically active enzyme together with its
bound coenzyme and/or metal ions.
Apoenzyme or apoprotein is the protein part of such an enzyme
Prosthetic group; A coenzyme or metal ion that is very tightly or
even covalently bound to the enzyme

Classification of enzymes
A)Recomended name: Many enzymes have been named by
adding the suffix -ase to the name of their substrate
(Glucosidase) or to a word or phrase describing their activity
(lactate dehydrogenase).
Some enzymes retain their original trivial (trypsin and
pepsin)
A)Systematic name; International Union of Biochemist (IUB)
system divides enzymes into six functional classes, each with
subclasses, based on the type of reaction catalyzed

No.

Class

Type of reaction catalyzed

1)

Oxidoreductases

Transfer of electrons (hydride ions or H atoms)

2)

Transferases

Group transfer reactions

3)

Hydrolases

Hydrolysis reactions (transfer of functional

groups to water)

4)

Lyases

Addition of groups to double bonds, or formation

of double bonds by removal of groups

5)

Isomerases

Transfer of groups within molecules to yield

isomeric forms

6)

Ligases

Formation of COC, COS, COO, and CON bonds

by condensation reactions coupled to ATP
cleavage

 Each enzyme is assigned a four-part classification number

and a systematic name, which identifies the reaction it
catalyzes
ATP + D-glucose

ADP + D-glucose 6- phosphate

Glucose phosphotransferase (E.C. number; 2.7.1.1)

2 = class name (transferase)
7 = subclass (phosphotransferase)
1 = phosphotransferase with a hydroxyl group as acceptor
1 = D-glucose as the phosphoryl group acceptor

HOW ENZYMES WORK

1) Enzymes provide an alternate, energetically favourable reaction
pathway different from the uncatalyzed reaction
2)

Enzyme active site chemically facilitates catalysis

Substrate is converted in a
product through an
intermediate state
(Transition state). Energy
required to reach the
transition state defines the
rate of reaction.

Enzyme Kinetics
Enzymes accelerate reactions by lowering the free energy
of activation
Enzymes do this by binding the transition state of the
reaction better than the substrate
Is the investigation of how enzymes bind substrates and
turn them into products.
Reactants and products conc. are usually 100 to 1000 times
greater than enzyme conc.
Enzyme-substrate complex (ES);
Transition state (ES*);
Enzyme-Product complex (EP)
Product (P) and free enzyme (E)
E+S

ES

ES*

EP

P+E

Factors affecting enzyme activity

Substrate concentration
Maximal velocity
Hyperbolic shape of the enzyme kinetics curve

Temperature
Increase of velocity with temperature
Decrease of velocity with higher temperature

pH
The ionization of the active site
enzyme denaturation

The Michaelis-Menten Equation

Michaelis and Menten's theory
Quantitative description of the relationship between rate of
enzyme-catalyed reaction (V), the concentration of substrate (S),
and two constants Vmax and Km
It assumes the formation of an enzyme-substrate complex
It assumes that the ES complex is in rapid equilibrium with free
enzyme
Breakdown of ES to form products is assumed to be slower than
(1) formation of ES and
(2) breakdown of ES to re-form E and S

The dual nature of the Michaelis-Menten equation

Combination of zero-order and 1st-order kinetics

k1

E+S

ES
k-1

k2

E+P
(k-2 is insignificant early in rxn)

Vo = k2 [ES]

[E] = ([Etotal] - [ES]

Rate of ES formation = k1 [E][S] = k1 ([Etotal] - [ES]) [S]
Rate of ES breakdown = k-1 [ES] + k2 [ES]
k1

([Etotal] - [ES]) [S] = k-1 [ES] + k2 [ES]

(steady state assumption)

k1 [Etotal][S] - k1[ES][S] = ( k-1 + k2 )[ES]

k1 [Etotal][S] = (k1[S] + ( k-1 + k2 )[ES]

[ES] =

[Etotal][S]

________________________

[Etotal][S]

____________

KM + [S]

[S] + (k2 + k-1 )

___________

k1

Vo = k2 [ES]

Vo =

k2 [Etotal][S]
____________

KM + [S]
Vo = Vmax when [Etotal] = [ES]
(at saturation)
Vo =

Therefore Vmax = k2 [Etotal]

Vmax[S]

____________

KM + [S]

The dual nature of the

Michaelis-Menten equation
Vo =

Vmax[S]
_________

Km + [S]

Combination of zero-order and first-order kinetics

When [S] is low, the equation for rate is first order in [S]

When [S] is high, the equation for rate is zero-order in [S]

The Michaelis-Menten equation describes a rectangular hyperbolic

dependence of Vo on [S]

Enzyme Kinetics: Michaelis-Menton Equation

Vo =

Vmax[S]

____________

KM + [S]

KM = [S]
when Vo =

Vmax

_____

2
From Lehninger
Principles of Biochemistry

The following data were obtained in a study of an enzyme known to follow

Michaelis Menten kinetics:

Km is the substrate
concentration that
corresponds to Vmax
2

V0
Substrate added
(mmol/min)
(mmol/L)

216
0.9
323
2
435
4
489
6
647
2,000

Calculate the Km for this enzyme.

Without graphing
Vmax = 647
Vmax /2 = 647 / 2 = 323.5
Km = 2 mmol/L

Understanding Km
Km is a constant
Km is a constant derived from rate constants
Km is, under true Michaelis-Menten conditions,
an estimate of the dissociation constant of E
from S
Small Km means tight binding; high Km means
weak binding
Enzyme

Substrate

Km (mM)

Glutamate dehydrogenase

NH4+
Glutamate
CO2

57
0.12
12

Carbonic anhydrase

Understanding Vmax
The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but
it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is
increased

Use linear plot and intercepts to

determine Km and Vmax

1
______

Vo

KM

+ ______
Vmax[S]
Vmax

_______

Double-Reciprocal or Lineweaver-Burk Plot

From Lehninger
Principles of Biochemistry

The turnover number

(also known as the molecular activity of the enzyme)

A measure of its maximal catalytic activity

kcat, the turnover number, is the number of substrate
molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate.
If the M-M model fits, k2 = kcat
kcat = Vmax/Et

Values of kcat range from less than 1/sec to many millions per sec

Turnover number comparison

Catalase
40,000,000 sec-1
Lysozyme
0.5 sec-1

Catalytic efficiency of an enzyme

Name for kcat/Km
An estimate of "how perfect" the enzyme is
kcat/Km is an apparent second-order rate constant
It measures how the enzyme performs when S is low
Catalytic efficiency cannot exceed the diffusion limit - the
rate at which E and S diffuse together
WT and a mutant protein kcat/Km comparision
WT sulfite oxidase

1.1

Mutant R160K

0.015

Enzyme Inhibitors
Reversible versus Irreversible
Reversible inhibitors interact with an enzyme via
noncovalent associations
Irreversible inhibitors interact with an enzyme via covalent
associations

Classes of Inhibition
Two real, one hypothetical
Competitive inhibition - inhibitor (I) binds only to E, not to ES
Uncompetitive inhibition - inhibitor (I) binds only to ES, not to
E. This is a hypothetical case that has never been
documented for a real enzyme, but which makes a useful
contrast to competitive inhibition
Noncompetitive (mixed) inhibition - inhibitor (I) binds to E and
to ES

Inhibitor (I) binds only to E, not to ES

Inhibitor (I) binds only to ES, not to E.

This is a hypothetical case that has
never been documented for a real
enzyme, but which makes a useful
contrast to competitive inhibition

Inhibitor (I) binds to E and to ES.

Enzyme Inhibition
From Lehninger
Principles of Biochemistry

Competitive
Inhibition
Kmchanges
while Vmax
does not

Uncompetitive
Inhibition
Km and Vmax
both change

Noncompetitive
(Mixed) Inhibition
Km and Vmax
both change
From Lehninger
Principles of Biochemistry

Succinate dehydrogenase is a classic example of competitive inhibition

Malonateisastrong
competitiveinhibitorof
succinatedehydrogenase
From Lehninger
Principles of Biochemistry

Competitive Inhibition
Kmchanges while Vmax
does not
+I
1/V
No I
-1 / Km
-1 / Kmapp

1/[S]

Where Kmapp = Km

= 1 + [I]
KI

Uncompetitive inhibition

1/V

/Vmax

+I
No I

- / Km

/Vmax
1/[S]
-1 / Km

= 1 + [I]
KI

Example of competitive inhibitors

Statin drugs [atorvastatin (Lipitor) and simvastatin)];
Cholesterol synthesis is catalyzed by
hydroxymethylglutaryl Co A reductase {HMG Co A
reductase }
antihyperlipidemic agents competitively inhibits the first
committed step in cholesterol synthesis thereby lowering
plasma cholesterol levels

Effects of Inhibitors on the parameters of Michaelis-Menten Equation

Type of inhibition

Vmaxapp

KMapp

No inhibitor

Vmax

KM

Competitive

Vmax

KM

Uncompetitive

Vmax/

KM/

Noncompetitive (Mixed)

Vmax/

KM/

= 1 + [I]

= 1 + [I]

KI

K I

Regulation of enzymatic activity

Two ways that this may occur:

Control of enzyme availability

Depends on rate of enzyme synthesis & degradation

Control of enzyme activity

Enzyme-substrate binding affinity may vary with binding
of small molecules called allosteric effectors (ex: BPG for
Hb)
Allosteric mechanisms can cause large changes in
enzymatic activity

Regulatory Enzymes
important in controlling flux through metabolic pathways

1. Allosteric enzymes

2. Regulation by covalent modification

From Lehninger
Principles of Biochemistry

Regulation by Feedback Inhibition

Conversion of L-threonine to Lisoleucine catalyzed by a sequence
five enzymes, E1-E5
L-isoleucine is an inhibitory allosteric
modulator of E1

From Lehninger
Principles of Biochemistry

Enzymes in diagnosis
Generally enzymes are present in cells in a higher conc than body fluids
Enzymes in serum is an indication of tissue or cellular damage
Commonly assayed enzymes are the amino transferases
Alanine aminotransaminase (ALT)
Aspartate aminotransferase (AST)
Lactate dehydrogenase (LDH)
Creatinine kinase (CK)
Gammaglutamyl transpeptidase (GGT)

Home work
Enzyme
Alkaline Phosphatase
Acid Phosphatase
Gammaglutamyl
transpeptidase (GGT)
Alanine
aminotransaminase
(ALT)
Aspartate
aminotransferase
(AST)
Lactate
dehydrogenase (LDH)
Creatinine kinase (CK)

Principal tissue

Normal level

Public health