This document discusses the principles and applications of enzymology. It begins with definitions of enzymes and their basic components and properties. It then covers topics like enzyme structure, kinetics, thermodynamics, techniques for studying enzymes, classification systems, and the specificity and mechanisms of enzyme catalysis. The document also examines controls on enzyme action, as well as applications like isolation and purification of enzymes. It concludes by discussing applications of enzymology in medicine and biotechnology, including enzyme engineering, immobilization, use in biosensors, and as part of bioelectrochemical cells.
2. 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
6. Definition
Enzymes are biological catalysts.
Enzyme components:
Active enzyme
or
Inactive protein (Apoenzyme) + Cofactor
[called: Holoenzyme]
Cofactor: Coenzyme (Organic molecule)
Or Metal
8. 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
9. 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
10. 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
12. 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
13. 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
14. 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
.
15. (2). Mild reaction conditions:
mostly, temperature < 100 o
C
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.
16. (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
.
20. 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
33. 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.
34. 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
39. 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.
40.
41. 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
42. 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.
43. Development of enzyme assay
Four criteria:
1). Absolute specificity
2). High sensitivity
3). High precision
4). Convenience and low cost
44.
45.
46. Methods for purification of enzymes
1. Centrifugation
2. Ion exchange,
3. Gel permeation chromatography,
4. Electrophoresis
5. Affinity chromatography,
6. Immunochemical techniques,
52. XII. Biotechnological applications of enzymes
Figure The protein engineering cycle.
The process starts with the isolation and
characterisation of the required enzyme.