------- enzymology --------

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 enzymologyII. The structure of enzymesIII. Specificity of enzyme actionIV. Bioenergetics of enzyme actionV. Investigation of enzyme active site structureVI. Chemical nature of enzyme catalysisVII. Kinetics of enzymatic reactionVIII. Enzyme inhibitionIX. Control of enzyme actionX. Enzyme isolation and purification XI. Application of enzymology in medicineXII. 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 classificationA. 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 [ES] Maximum velocity Vmax

Machaelis constant Km

BiocatalystsEnzymes: 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; (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) 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)