酵素學原理與應用
(Principles and Application of Enzymology)
潘榮隆
(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
酵素: 脆弱、敏感、而又美麗的生命組成分子
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 氧化還原酵素
2) Transferase 轉移酵素
3) Hydrolase 水解酵素
4) Lyase 裂解酵素
5) Isomerase 異構轉化酵素
6) Ligase 連接酵素
New set of notation and terminology
Reactant -----> Substrate [S]
Catalyst -----> Enzyme [E]
Product -----> Product [P]
Enzyme-substrate complex
Maximum velocity
Machaelis constant
[ES]
Vmax
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
catalyze this reaction.
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
1
Km
1
1
---- = ------ x ------ + -----v
[S]
Vmax Vmax
Eadie-Hofstee
v
Vmax
v
---- = ------- - -----[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;
(2). conformational changes caused by
regulators
(3). specific inhibitor macromolecules
(4). availability of substrate or cofactor
(5). product inhibition
(6). non enzyme-catalyzed reactions
(Cont.)
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
maximum
possible purity.
catalytic
activity,
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) 酵素固定化(Immobolized enzymes)
XII.-(c)生物感應器 (Biosensor)
生物感測器生物分子辨認元件之組成份子
XII.-(d)生物電池 (Bioelectrochemical cell)