Chapter Outline

I. Introduction
Enzyme

i.

Biologic proteins that catalyze biochemical

reactions
ii. Not consumed or changed in composition
iii. Found in all body tissue (intracellular) and is
in serum after cell injury

Chapter Outline
I. Introduction

Function of Enzymes
i.
ii.
iii.
iv.
v.
vi.

Hydration of Carbon Dioxide (respiration)

Nerve Induction
Muscle Contraction
Nutrient Degradation (Digestion)
Growth and Reproduction
Energy Storage and Use

Enzymes
II. General Properties and
Definitions
A. Components of an Enzyme
i.

Active Site
A cavity of an enzyme where
substrates bind and undergo a
chemical reaction.
ii. Allosteric Site
A cavity other than the active site
that binds regulatory (effector)
molecules.

Enzymes
II. General Properties and
Definitions
A. Components
Allosteric
promoter

of an

Allosteric
Enzyme Inhibitor

Enzymes

II. General Properties and

Definitions
B. Terms associated with enzymes
i. Substrates
ii. Cofactors
iii. Isoenzyme
iv. Apoenzyme
v. Holoenzymes
vi. Proenzyme or Zymogens
vii.Allosteric enzymes
viii.Inhibitors

Enzymes
II. General Properties and
Definitions
B. Terms associated with enzymes
i.

Substrates
Substances acted upon enzymes
Specific for each of their particular
enzyme

Enzymes
II. General Properties and Definitions
B. Terms associated with enzymes
ii. Cofactors
Non protein substances added in the enzyme
substrate complex to manifest the enzyme
activity
a. Coenzyme or Prosthetic group
An organic cofactor
Nucleotide (E.g. NAD, NADP) and Vitamins
b. Activator
An inorganic cofactor
Metal ion (E.g. Cl-1, Mg++,Cu+)

Enzymes
II. General Properties and
Definitions
B. Terms associated with enzymes
iii. Isoenzyme
Similar enzymatic activity but differ in
physical, biochemical and immunologic
characteristics
iv. Apoenzyme
The protein portion of the enzyme
Subject to denaturation, in which
enzyme losses its activity

Enzymes
II. General Properties and
Definitions
B. Terms associated with enzymes
v. Holoenzyme
An active substance formed by
combination of a co-enzyme and an
apoenzyme.
vi. Proenzyme or Zymogens
An inactive enzyme precursor
E.g. Coagulation factors and digestive
enzymes

Enzymes
II.

General Properties and Definitions

B. Terms associated with

enzymes
vii.Allosteric enzymes
.
Regulator of cellular
processes, but not all enzymes
are allosteric.
.
Some can be allosteric
provided that they are composed
of quaternary structures with
two or more protein chain
containing the active sites

Enzymes
Two kinds of allosteric enzymes:
1. Homoallostery. This is a
cooperative substrate binding and
activation wherein substrate is a
homotropic effector.
Therefore the binding of substrate to
one active site alters the substrate
binding affinity and/or catalytic
activity at other active sites on the
multimeric enzyme.

Enzymes
Two kinds of allosteric enzymes:
2. Heteroallostery. This merely involves the
regulation byheterotropic effector molecules,
which can be positive (activation) or
negative (inhibition).
These heterotropic effectors usually bind at a
site other than the active site.
These effectors can activate or inhibit the
activity of an enzyme..

Enzymes
viii. NHIBITORS OF ENZYMATIC
REACTIONS
An
inhibitor is any compound that reduces the velocity
of the enzyme-catalyzed reaction when present in
the reaction mixture.
Penicillin irreversibly (covalently) inhibits an enzyme
involved in bacterial cell wall synthesis
.

Ibuprofen and many other nonsteroidal

antiinflammatory drugs (NSAIDs) are reversible
competitive inhibitors of the cyclooxygenase activity of
prostaglandin H2 synthase.

Enzymes
Inhibitors that occupy the active site and
prevent a substrate molecule from binding
to the enzyme are said to be active sitedirected (or competitive, as they
'compete' with the substrate for the
active site).
Inhibitors that attach to other parts of the
enzyme molecule, perhaps distorting its
shape, are said to be non-active sitedirected (or non competitive

KINDS OF INHIBITORS
1. Competitive Inhibition
In competitive inhibition, a chemical
inhibitor competes for the active site with
the substrates.
The reaction on the active sites depends
upon the affinity of the enzyme for the
substrate and for the inhibitor.
Often, the enzyme has a greater affinity for
the inhibitor than it does for the substrate.

Examples of competitive
inhibitors
In case of Methanol poisoning, it occurs because
methanol is oxidized to formaldehyde and formic
acid which attack the optic nerve causing blindness.
Ethanol( an example of competitive inhibitor) is
given as an antidote for methanol poisoning because
ethanol competitively inhibits the oxidation of
methanol.
It is shown when ethanol is oxidized in preference to
methanol.
Consequently, the oxidation of methanol is slowed
down so that the toxic by-products do not have a
chance to accumulate.

Inhibitors
Noncompetitive Inhibition
It is a substance that interacts with
the enzyme, but usually not at the
active site.
It reacts either remote from or very
close to the active site.

Inhibitors
Noncompetitive Inhibition
The net effect of a noncompetitive
inhibitor is to change the shape of
the enzyme and thus the active site,
The substrate can no longer interact
with the enzyme to give a reaction.

Inhibitors
Noncompetitive Inhibition
One good example of noncompetitive
inhibitor is the nerve gases such as
diisopropylfluorophosphate (DFP)
This inhibits the active site of
acetylcholine esterase by reacting
with the hydroxyl group of serine to
make an ester.

Chapter Outline
III.Enzyme Classification and
Nomenclature
1.
2.
3.
4.
5.
6.

Oxidoreductases
Transferase
Hydrolases
Lyases
Isomerases
Ligases

Enzyme Classification and

Nomenclature
The 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 corresponding meaning.

Enzyme Classification and

Nomenclature
E.C. 1.1.1.1
Alcohol:NAD+oxidoreductase

The first number shows to which of the six
main divisions (classes) the enzyme belongs,
The second figure indicates the subclass,
The third figure gives the sub-subclass,
Tthe fourth figure is the serial number of
the enzyme in its sub-subclass.

ENZYME NOMENCLATURE
CLASS

RECOMMEND ABBREVIA
ED NAME
TED NAME

E.C
SCIENTIFIC
CODE NAME
NO.

1. Oxido
reductase

Lactate
dehydrogenas
e

LDH

1.1.1.
27

2.
Transferase

2.1 Aspartate
amino
transferase

SGOT
2.6.1.
( Serum
1
Glutamate
Oxaloacetat
e
transamina
se)

L-Aspartate ,2oxaloglutarate
Amino
transferase

2.2 Alanine
amino
transferase

SGPT
( Serum
Glutamate
Pyruvate
transamina

L-Alanine, 2oxaloglutarate
amino
transferase

2.6.1.
2

L-Lactate NAD+
oxidoreductase

ENZYME NOMENCLATURE
CLASS

RECOMME
NDED
NAME

ABBREVIAT E.C
ED NAME
CODE
NO.

SCIENTIFIC
NAME

3.
Hydrolases

Alkaline
Phosphatas
e

ALP

3.1.3.1

Orthophosphoric,
monoester
phosphohydrolas
e (alkaline
optimum)

Acid
Phosphatas
e

ACP

3.1.3.2

Orthophosphoric,
monoester
phosphohydrolas
e (acid optimum)

-Amylase

AMS

3.2.1.1

1,4- D- Glucan,
Glucanohydrolas
e

Enzymes
III.Enzyme Classification and
Nomenclature
1. Oxidoreductases

Catalyze redox reaction between two substrates

A- + B A + B E.g: Dehydrogenase (Lactate Dehydrogenase)

Enzymes
III.Enzyme Classification and
Nomenclature
2. Transferases

Catalyze the transfer of a group (Phosphate,

methyl, etc.) between two substrates (A-X + B
A + B-X)
E.g: Transferase (ALT, AST, GGT) and Kinase
(CK)

Enzymes
III.Enzyme Classification and
Nomenclature
2. Transferases

Catalyze the transfer of a group (Phosphate,

methyl, etc.) between two substrates (A-X + B
A + B-X)
E.g: Transferase (ALT, AST, GGT) and Kinase
(CK)

Enzymes
III.Enzyme Classification and Nomenclature
3. Hydrolases

Catalyze hydrolysis of various bonds

AB + H2O AOH + BH
E.g: Amylase (AMY), Lipase (LPS), Phosphatase (ALP, ACP)

Enzymes
III.Enzyme Classification and
Nomenclature
4. Lyases

Catalyze the removal of groups from

substrates without hydrolysis; the product
remain double bonds
ATP cAMP + PPi
Fructose biphosphate aldolase (ALS)

Enzymes
III.Enzyme Classification and
Nomenclature
4. Lyases

Catalyze the removal of groups from

substrates without hydrolysis; the product
remain double bonds
ATP cAMP + PPi
Fructose biphosphate aldolase (ALS)

ENZYME MECHANISMS
1. Lowering the activation energy. It is done
by creating an environment in which the
transition state is stabilized
Example is the straining the shape of a
substrateby binding the transition-state
conformation of the substrate/product
molecules,
The enzyme distorts the bound substrate(s) into
their transition state form,
Thereby reducing the amount of energy
required to complete the transition).

LOWERING ACTIVATION
ENERGY

1. Increase the proximity of the reactants,

2. Increase the concentration of the reactants,
3. Increase the surface area of the reactants
4. Increase the temperature of the reactants,
5. Use a catalyst (a substance which speeds up
a chemical reaction but is not used up),
6. Use an enzyme.

ENZYME MECHANISMS
2. Providing an alternative
pathway.
Temporarily reacting with the substrate
to form an intermediate Enzymesubstrate (ES) complex, which would be
impossible in the absence of the
enzyme.

ENZYME MECHANISMS
3, Reducing the reaction entropy
change.
Bringing substrates together in the correct
orientation to react.
Considering enthalpy change (H ) alone
overlooks this effect.
The entropic effect involves
destabilization of the ground state, and its
contribution to catalysis is relatively small

MODELS OF ENZYME ACTION

1.Lock and key

hypothesis

2. Induced Fit Hypothesis

. The change in shape is 'induced' by the approaching substrate

molecule. This more sophisticated model relies on the fact that
molecules are flexible because single covalent bonds are free
to rotate.

CHARACTERISTICS AND PROPERTIES OF ENZYMES

Enzymes
Enzyme Kinetics
Catalytic Mechanism of Enzymes
i.

Absolute specificity
Combines with only one substrate and
catalyzes only one reaction (E.g. CK, LD)
ii. Group specificity
Combine with all substrates containing a
particular chemical group (E.g. ACP, ALP)
iii. Bond specificity
Specific to chemical bonds (E.g. AMY, LPS)
iv. Sterioisometric specificity
Combine with one optical isomer (E.g. LDH,
G6PD)

Enzymes
IV.Enzyme Kinetics
i.

Catalytic Mechanism of Enzymes

Enzymes catalyze physiologic reactions by

lowering the activation energy level that
the reactants must reach

Enzymes
IV.Enzyme Kinetics
i.

Catalytic Mechanism of Enzymes

i.

Relationship between Enzyme, Substrate

and Product

Enzymes
IV.Enzyme Kinetics
i.

Catalytic Mechanism of Enzymes

i.

Relationship between Enzyme, Substrate

and Product

Order of Reaction
the order of the reaction can be
specified in terms of the order with
respect to each specific reactant
or the overall order of the
reaction.
Consider the reaction mA +
nB<===> C.
The rate equation is R = k[A]m[B]n.

Order of Reaction
Consider the reaction mA + nB<===> C.

The rate equation is R = k[A]m[B]n.

If the exponent m in the equation is 1, then the

reaction is said to be First order with respect to A.
If m = 2, In then, 2A + 1B <===> 1C) then it is said
to be second order with respect to A and first order
with respect to B.
Now, if m = 1 and n = 1, since it is first order with
respect to A and B, then the overall order of the
reactions said to be Second order (or m + n).

HALF -LIFE
HALF-LIFE
By definition, Half-life (t1/2) is the time required
for half of the original concentration of the
limiting reactant to be used up as the reaction
takes place or half-life is equal to 0.69 /K.
Thus, the larger the rate constant (K), the
faster will deplete the substrate.
As noted, in a first order reaction, the half-life
is inversely proportional to the rate
constant (k).

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions
1.
2.
3.
4.
5.
6.

Substrate Concentration
Enzyme Concentration
pH
Temperature
Cofactors
Inhibitors

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions
1. Substrate Concentration
First order kinetics (Michaelis- Menten
hypothesis)
Reaction rate is proportional to the
substrate concentration.

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions
2. Enzyme Concentration
Zero-order kinetics
Only a fixed number of substrate (in
excess) is converted to product per
second

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions

Substrate and Enzyme Concentration

First order and Zero Order kinetics

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence
Enzymatic Reactions
3. pH
Common range 7.0-8.0
Controlled by buffers
4. Temperature
Within 0.1C
Enzyme is active
at 25C, 30C,
37C.

Optimum pH of Different Enzymes

Enzymes
Sucrase
Ribonuclease

Sources
Intestine
Pancreas

Glucosidase
Acetylcholine
sterase
Enolase

Yeast

Arginase
Pepsin

Erythrocytes

Substrates
Sucrose
3-50Cytidylyl
adenine
Methyl--Dglucoside
Acetylcholine

Optimum pH
6.2
7.0
5.4
7.5

Rabbit Muscle 2-Phospho-D- 6.8

Glycerate
Beef Liver
L-Arginine
8.4-9.7
Gastric
Acetyl L1.5-2.5
mucosa
Phenylalanine
, Lphenylalanine

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions
5. Cofactors
Activators: Metalic (Ca2+) and Non Metallic
(Cl- )
Coenzymes (prosthetic groups): 2nd
substrates (NAD)

Chapter Outline
IV.Enzyme Kinetics
ii. Factors that Influence Enzymatic
Reactions
6. Inhibitors

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

Measurement of catalytic activity

1. in product concentration
2. in substrate concentration
3. or in coenzyme concentration
(NADH)
4. in altered enzyme concentration

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

Measurement of catalytic activity

1. in product concentration
2. in substrate concentration
3. or in coenzyme concentration
(NADH)
4. in altered enzyme concentration

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

Measurement of catalytic activity

a. Dependent on enzyme concentration
b. Performed in zero-order kinetics (linear
phase)

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

General methods of measuring

enzymatic reaction
1. Fixed time (Two point) Assay
2. Continuous-monitoring or kinetic
assays

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

General methods of measuring

enzymatic reaction
1. Fixed time (Two point) Assay
Reagents are combined and the
amount of reaction is measured
(AMS, LPS, ACP, ALP)

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

General methods of measuring

enzymatic reaction
1. Fixed time (Two point) Assay
Reagents are combined and the
amount of reaction is measured.

Chapter Outline
IV.Enzyme Kinetics
iii. Measurement of Enzyme Activity

General methods of measuring enzymatic

reaction
2. Continuous-monitoring or kinetic
assays
Measurements at specific time
intervals
Rate of change in substrate, cofactor,
product.

Chapter Outline
IV.Enzyme Kinetics
iv. Calculation of Enzyme Activity
1. IU (EC)

Amount of enzyme that will catalyze the

reaction of 1 mol of substrate per
minute (mol /min)

2. Kat (SI)

Amount of enzyme that will catalyze the

reaction of 1 mol of substrate per second
(mol/s)

Chapter Outline
IV.Enzyme Kinetics
v. Measurement of Enzyme Mass

Immunoassays
Electrophoresis