(Principles and Application of Enzymology)

(Rong Long Pan, Rev., Ph.D., & M.Div.)

 Outlines
I. Introduction to enzymology
II. The structure of enzymes
III. Specificity of enzyme action
IV. Bioenergetics of enzyme action
V. Investigation of enzyme active site structure
VI. Chemical nature of enzyme catalysis
VII. Kinetics of enzymatic reaction
VIII. Enzyme inhibition
IX. Control of enzyme action
X. Enzyme isolation and purification
XI. Application of enzymology in medicine
XII. Biotechnological applications of enzymes
 :
 Definition

Enzymes are biological catalysts.

Enzyme components:
Active enzyme
or
Inactive protein (Apoenzyme) + Cofactor
[called: Holoenzyme]

Cofactor: Coenzyme (Organic molecule)

Or Metal
(1) Enzyme properties
(a) Physical properties
(b) Chemical properties

(2) Structure

(3) Kinetics

(4) Thermodynamics

(5) Biological properties

3. Basic techniques:
A. General technique:
1. Potentiometry, 2. Spectrophotometry,
3. Centrifugation 4. Ion exchange,
5. Gel permeation chromatography,
6. Electrophoresis
7. Affinity chromatography,
8. Radiochemistry,
9. Immunochemical techniques,
10. Protein purification,
11. Other non-conventional biochemical
techniques
B. Enzyme Kinetics:

C. Spectroscopy for enzymology:

1. UV/Vis spectrophotometry,
2. IR, Raman spectrophotometry,
3. CD and ORD,
4. Fluorescence and phosphorescence,
5. ESR and NMR,
6. Electron microscopy,
7. X-ray and neutron diffraction
 Enzyme classification
A. Organizations handle the enzyme classification:
International Union of Biochemistry
International Union of Pure and
Applied Chemistry.

B. Name of enzymes:
(a) Name of substrate + (b) -ase at end of words.

The Enzyme Commission (1961) offered

code numbers (EC) with four elements
(1)First figure: Six main classes:

1) Oxidoreductase
2) Transferase
3) Hydrolase
4) Lyase
5) Isomerase
6) Ligase
New set of notation and terminology

Reactant -----> Substrate [S]

Catalyst -----> Enzyme [E]

Product -----> Product [P]

Enzyme-substrate complex [ES]

Maximum velocity Vmax

Machaelis constant Km
 Biocatalysts
Enzymes: proteins with catalytic
activity
Ribozyme:
fragment of RNA can also act as
catalyst for reaction involving
hydrolysis of RNA.

Abzyme:
antibody which binds the complex
of transition state of an reaction can
III. Specificity of enzyme action

(1). High reaction rate:

106~1012 higher, even 1014 higher.

rate ratio with/without enzyme

eg. hexokinase > 1010
phosphorylase > 3 x 1011
alcohol DHase > 2 x 108
urase kinase > 104.
(2). Mild reaction conditions:
mostly, temperature < 100 oC
atmospheric pressure, neutral pH.
eg. N2 --------> NH3
[1]. Nitrogenase, at 300 K, neutral pH,
requires ATP.

[2]. Industrial (Harber) method:

N2 + H2 --------> NH3
at 700~900 K,
1100~900 atmospheric pressure,
catalyst: Fe, oxides of other metals.
(3). High specificity, less side effects
[1]. Protein synthesis by ribosome:
1,000 a.a. polypeptide, no error.
Chemical protein synthesis:
~50 a.a. oligopeptide, many errors.
[2]. Group specificity
[3]. Absolute or near absolute specificity
(a). Stereochemistry
(b). Proof-reading system:
DNA or protein synthesis,
1 mutation/108~1010.
IV. Bioenergetics of enzyme action
V. Active site structure of enzyme
 Active site studies
Identification of active site
Trapping the enzyme-substrate complex
The use of substrate analogue
Modification of active site residues
Modification by protease
Modification by site-directed mutagenesis
Effect of pH, etc
Substrate analogue Chemical modification
Mechanism of enzyme action
VI. Chemical nature of enzyme catalysis

Mechanism for
enzyme specificity
V. Kinetics of enzymatic reaction
Enzyme

A ---------------- B
 Michaelis-Menten reaction
k1 k2
E + S ------ ES ----- E + P
k-1

Vmax [S]
v = ----------------
Km + [S]
Lineweaver-Burke Eadie-Hofstee
1 Km 1 1 v Vmax v
---- = ------ x ------ + ------ ---- = ------- - ------
v [S] Vmax Vmax [S] Km Km
VI.
Kinetics of enzyme inhibition

Competitive inhibition
Non-competitive inhibition
 IX. Control of enzyme action

If no control, all metabolic process would

become equilibrium with surroundings.

Two ways of control:

(1). amount of enzyme, synthesis and
degradation; long term response to
environment;
(2). activity of enzyme already
presented in the cell.
 Control of activity of single enzymes

(1). change in covalent structure of an

enzyme; (Cont.)

(2). conformational changes caused by

regulators
(3). specific inhibitor macromolecules
(4). availability of substrate or cofactor
(5). product inhibition
(6). non enzyme-catalyzed reactions
Covalent modifications of enzymes:

Reversible vs Irreversible

(1). Phosphorylation
(2). ADP-ribosylation
(3). Adenylylation
(4). Methylation
(5). Acetylation
(6). Tyrosinolation
(7). Sulphation
Feedback control of metabolism
Cooperative control of enzyme
 X. Enzyme isolation and purification

Strategy to Enzyme Purification

Highly Purified protein is very important to

biochemical and biophysical studies of
proteins.

To gain maximum catalytic activity,

maximum
possible purity.
2. Basic steps of purification:
a). Development of suitable assay
procedures
b). Selection of material sources
c). Solubilization of desired molecules
d). Stabilization of molecules
repeatedly at each steps
e). Development of a series of isolation

and concentration procedures

Choice of methods depends on:

1). Scale of the preparation and the

yield of enzyme required.
2). Time availabile for the preparation.
3). The equipment and expertise
available in lab.
 Development of enzyme assay

Four criteria:
1). Absolute specificity

2). High sensitivity

3). High precision

4). Convenience and low cost

Methods for purification of enzymes
1. Centrifugation
2. Ion exchange,
3. Gel permeation chromatography,
4. Electrophoresis
5. Affinity chromatography,
6. Immunochemical techniques,
XI. Application of enzymology in medicine
XII. Biotechnological applications of enzymes

Figure The protein engineering cycle.

The process starts with the isolation and
characterisation of the required enzyme.
XII.-(a)
XII.-(b) (Immobolized enzymes)
XII.-(c) (Biosensor)

XII.-(d) (Bioelectrochemical cell)