Chapter 6. Enzymes
I.
II.
III.
IV.
An introduction to enzymes
Enzyme kinetics (酶促反应动力学)
Enzyme mechanism (酶的作用机制)
Introduction to enzyme engineering
(酶工程简介)
1
I. An introduction to enzymes
1.
2.
3.
4.
Importance of enzymes (酶的重要性)
The history of enzyme research (酶研究的历史)
Enzyme properties (酶的性质)
Classification and nomenclature (酶的分类和命
名)
5. Enzyme activity and its determination (酶活性
及其测定)
6. Separation, purification and storage of enzymes
(酶的分离提纯和储存)
2
1. Importance of enzymes
1) Extraordinary catalytic power
2) Central to all biochemical processes
3) Practical importance
I. Introduction
In routine life
In industry
In medical care
……
C6H12O6 + 6 O2  6 CO2 + 6 H2O + 2870 kJ/mol
Enzymes: Kinetic control over thermodynamic potentiality
3
4
Prof. K. Barry Sharpless: Winner of the
Nobel Prize in Chemistry, 2001
"for his work on chirally catalysed oxidation reactions"
“The secret life of enzymes”
5
2. The history of enzyme research
When?
8000 yrs ago
What?
Brew fermentation
Who?
Chinese
1850s
Louis Pasteur
1878
1894
Fermentation of sugar into
alcohol by yeast
“Enzyme”
“Lock & key” theory
1897
1913
Fermentation with yeast extract
Michaelis-Menten equation
Büchner brothers
Michaelis &
Menten
1926
Isolation and crystallization of
urease
Cell-free fermentation
James Sumner
1907 Nobel
Kühne
Fisher
Eduard Büchner
6
Nobel Prize in Chemistry
When?
What?
Who?
1946
Crystallization and preparation of
enzymes
Sumner, Northrop, Stanley
1957
nucleotides and nucleotide co-enzymes
Todd
1972
Structure & activity of RNase
Anfinsen, Moore, Stein
1975
Stereochemistry of enzyme-catalyzed
reactions
Cornforth
1989
Catalytic properties of RNA
Altman & Cech
Enzymatic mechanism of ATP synthesis
Boyer & Walker
First discovery of ion-transporting
enzyme, Na+, K+-ATPase
Skou
1997
7
Nobel Prize in Physiology/Medicine
When?
What?
Who?
1931
Respiratory enzyme
Warburg
1953
Co-enzyme A and its importance in
intermediary metabolism
Lipmann
1955
Oxidation enzymes
Theorell
1965
Genetic control of enzymes and virus
synthesis
Jacob, Lwoff,
Monod
1978
Restriction enzymes and their
application in molecular genetics
Arber, Nathans,
Smith
8
3. Enzyme properties
1) Most enzymes are proteins



Exhibiting all protein properties
Simple & conjugated enzymes
Mono-/oligo-/multimeric enzymes
I. Introduction
2) Enzymes are biocatalysts


Working as regular chemical catalysts
Advantages over chemical catalysts
9
Enzymes as proteins
P.323
Structure
Acid-base properties
Chemical/physical properties
Denaturation
Monomeric enzyme (单体酶)
Oligomeric enzyme (寡聚酶)
Multienzyme complex (多酶复合体)
Simple enzymes (简单酶)
Conjugated enzymes (复合酶)
Holoenzyme
= Apoenzyme + Cofactor
(全酶)
(脱辅酶)
(辅因子)
Active
Inactive
Coenzyme (辅酶)
-- e.g. NAD+, CoQ
-- Loosely bound
P.323-326
Prosthetic group (辅基)
-- e.g. FAD, metal ions
-- Tightly bound
10
Enzyme Cofactors
NAD+
(Oxidized form)
NADH + H+
(Reduced form)
O
OH
H3C
C
H
H3C
COOH
Lactate dehydrogenase
(乳酸脱氢酶)
I. Introduction
Lactate
(乳酸)
C
COOH
Pyruvate
(丙酮酸)
Nicotinamide Adenine Dinucleotide (烟酰胺腺嘌呤二核苷酸)
NAD+ + 2e- + 2H+  NADH + H+
11
12
13
Enzymes as biocatalysts
a). Working as chemical catalysts
[P]
S  P Equilibrium constant K’eq =
[S]
S  P Reaction rate V = k [S]
k is a first-order rate constant
(一级速度常数)
S1 + S2  P Reaction rate V = k [S1][S2]
k is a second-order rate constant (二级速度常数)
T -∆G /RT
≠)：与速度常数 (k) 有关
活化能(
∆G
e
k=
h
-- 决定反应速度
≠
∆G’o = -RT ln K’eq
自由能变化(∆G’o)：与平衡常数 (K’eq)有关
-- 决定反应方向
14
How does an enzyme work?
Go
E + S  ES  EP  E + P
Use of an enzyme
Can make a reaction faster: G  k
Cannot change the direction of a reaction: Go unchanged  Keq unchanged
15
Activation Energy (活化能, kJ/mol)
Reaction
No catalyst
Chemical catalyst Enzyme
H2O2  H2O + O2
75.4
48.9
8.4
Hydrolysis of sucrose
1339.8
104.7
39.4
16
P.320-323
Enzymes as biocatalysts
b) Advantages over chemical catalysts
 High catalytic efficiency
Substrate specificity (底物专一性)
 High specificity
Enantioselectivity (旋光异构选择性)
 Mild conditions
Prochiral selectivity (潜手性选择性)
 Regulation
I. Introduction
Regioselectivity (区域选择性)
……
pH
Temperature
Pressure
By controlling enzyme concentration
By using hormones (激素)
By feedback inhibition (反馈抑制)
By inhibitors/activators (抑制剂/激活剂)
17
Catalytic power
Some rate enhancements produced by enzymes
I. Introduction
Cyclophilin
105
Carbonic anhydrase
107
Triose phosphate isomerase
109
Carboxypeptidase A
1011
Phosphoglucomutase
1012
Succinyl-CoA transferase
1013
Urease
1014
Orotidine monophosphate decarboxylase
1017
CO(NH2)2 + 2H2O + H+
Urease
2NH4+ + HCO3-
k = 3 x 10-10 s-1 (without enzyme)
k = 3 x 104 s-1 (with enzyme)
18
Enzyme specificity
P.332
O
Substrate specificity
(底物专一性)
Enantioselectivity
(旋光异构专一性)
C
尿素
H2N
R
NH2 + H2O
Urease (ëåø)
L-Amino acid
oxidase
H
C
COOH + H2O + O2
NH2
(L-amino acid)
COOH + NH3 + H2O2
C
R
O
CO2 + 2NH3
(-keto acid)
O
Regioselectivity
(区域选择性)
C
R1
Lipase (脂肪酶)
OR2 + H2O
R1COOH + R2OH
OH
OH
HOOC
Prochiral selectivity
(潜手性选择性)
H
C
延胡索酸水合酶
3
H2O
C
H
HOOC
H
H
COOH
COOH
3
H
延胡索酸
3
H
苹果酸
19
P.326
4. Classification and Nomenclature
(酶的分类和命名)
EC No.
1
A new system for enzyme nomenclature suggested
by the Enzyme Commission in 1961:
Class
Types of reactions catalyzed
Oxidoreductases
2
Oxidation-reduction reaction
(氧化还原酶)
(氧化还原反应)
Transferases (转移酶) Group-transfer reactions (转移基团)
3
Hydrolases (水解酶)
4
Lyases (裂合酶)
Group removal to form a double bond, or
group addition to a double bond
5
Isomerases (异构酶)
Isomerization reactions (异构反应)
6
Ligases (连接酶)
Hydrolysis reactions (水解反应)
Formation of bonds with ATP
cleavage (形成C-C, C-N, C-S等键)
Enzymes are classified by the reactions they catalyze.
20
Enzyme Classification and Examples (酶的分类举例)
Enzyme Class
Example
Reactions Catalyzed
Oxidoreductase Alcohol dehydrogenase CH3-CH2-OH + NAD+  CH3-CHO
+ NADH + H+
(氧化还原酶)
(乙醇脱氢酶)
Transferase
(转移酶)
Hexokinase (己糖激酶) Glucose + ATP  Glucose-6phosphate + ADP
Hydrolase
(水解酶)
Lipase (脂肪酶)
Lyase (裂合酶)
Pyruvate decarboxylase
(丙酮酸脱羧酶)
Isomerase
(异构酶)
Alanine racemase
(丙氨酸消旋酶)
Ligase
(连接酶)
Pyruvate carboxylase
(丙酮酸羧化酶)
R1-COOR2 + H2O 
R1-COOH + R2-OH
O
H3C
O
O
C
-
C
+ H+
H3C
O
C
H
+ CO2
D-Alanine  L-alanine
Glucose  fructose
O
H3C
C
O
C
O
-
O
C
H2
C
-
O
+ HCO3- + ATP
O
O
C
C
O-
+ ADP + Pi
21
Enzyme Nomenclature (酶的命名)
ATP + D-glucose
ADP + D-glucose 6-phosphate
Trivial name (俗名)
Glucokinase (葡糖激酶 )
ATP: D-glucose 6-phosphotransferase
(ATP: D-葡萄糖6-磷酸转移酶)
EC 2.7.1.2
Class: transferase
(转移酶)
Subclass: phosphotransferase
(磷酸转移酶)
Systematic name (系统命名)
Systematic number (系统编号)
Individual entry:
D-glucose as the
phosphoryl-group acceptor
(葡萄糖上的羟基是接受体)
Sub-subclass:
Phosphotransferase with
a –OH group as acceptor
(羟基是接受体)
22
Class 2: Transferases (转移酶类)
EC Number
Systematic name and subclasses
2
Transferases (transfer of functional groups)
2.1
Transferring C-1 groups
2.1.1
Methyltransferases
I. Introduction
2.2
2.3
2.4
Transferring aldehydic or ketonic residues
Acyltransferases
Glycosyltransferases
2.6
2.6.1
2.7
Transferring N-containing groups
Aminotransferases
Transferring P-containing groups
2.7.1
2.7.1.2
With an alcohol group as acceptor
With D-glucose as acceptor
23
5. Enzyme activity and its determination
(酶活力及其测定)
P.335
I. Introduction
1) Definition of enzyme activity (酶活性的定义)
 Activity (酶活力)
 Activity unit (酶的活力单位)
 Specific activity (比活力)
 Turnover number (转换数)
2) Determination of enzyme activity (酶活力的
测定)
24
P.335-336
Activity and activity unit
 Activity (酶活力)
-- Ability of the enzyme to catalyze a reaction
-- Usually expressed as the initial reaction rate
(通常以初始反应速度表示)
S  P, V = [P]/t = -[S]/t
 Activity unit (酶活力单位)
I. Introduction



at 25oC under optimal conditions:
 International unit: 1 IU = 1 mol/min
 Katal: 1 Kat = 1 mol/sec
Relationship between the two units:
1 Kat = 60 x 106 IU
Optimal reaction conditions (最佳反应条件):
pH, temperature, [S], buffer
25
An example for measuring enzyme activity:
O
H2N
C
尿素
NH2 + H2O
Urease
Product yielded
per min
Solution A:
1 ml
10 mol
Solution B:
0.1 ml
5 mol
Urease (ëåø)
CO2 + 2NH3
Reaction rate
Total activity
units
Activity units/
ml solution
10 mol/min
10 IU
10 IU/1 ml
= 10 IU/ml
5 IU
5 IU/0.1 ml
= 50 IU/ml
5 mol/min
The enzyme solution B is more active than the enzyme solution A.
26
Specific activity and turnover number
P.336  Specific activity (比活力):
 Number of enzyme units per milligram of total protein (每毫克蛋
白所含的酶活力单位数)
 A measure of enzyme purity (酶的纯度)
Total activity (U)
Specific activity (U/mg) =
Total protein (mg)
P.361  Turnover number (kcat, 转换数):
 Number of substrate molecules converted in a given unit of time
on a single enzyme molecule when the enzyme is saturated with
substrate (酶被底物饱和时每秒钟每个酶分子转换底物的分子数)
 A measure of the catalytic power of the enzyme (酶的催化效率)
No. of substrate molecules
Turnover Number (s-1) =
No. of enzyme molecules
/sec
27
An example for measuring specific activity:
Activity units/
ml solution
Urease
Solution A:
1 ml
Solution B:
0.1 ml
10 IU/ml
50 IU/ml
Protein content
Specific activity
1 mg/ml
10/1
= 10 IU/mg protein
0.1 mg/ml
50/0.1
= 500 IU/mg protein
Urease
Total activity units
Total protein
content
Specific activity
Solution A:
1 ml
10 IU
1 mg
10/1
= 10 IU/mg protein
Solution B:
0.1 ml
5 IU
0.01 mg
5/0.01
= 500 IU/mg protein
The purity of the enzyme is greater in Solution B than in Solution A.
28
P.336
Determination of enzyme activity
(酶活力测定)

What you need to know before the assay:
 The enzymatic reaction
 All the optimum reaction conditions:
pH, temperature, buffer, [S], cofactor, …

It is the initial rate of the enzymatic reaction that is
determined as the enzyme activity (测定酶促反应的
I. Introduction
初速度作为酶活性)

Activity assays:






Spectrophotometry (分光光度法)
Fluorometry (荧光法)
Electrochemical method (电化学法)
Detection of radioactive isotopes (放射性同位素测定法)
GC (气相色谱法)
HPLC (高效液相色谱法)
29
NAD+
(Oxidized form)
NADH + H+
(Reduced form)
O
OH
H3C
C
H
H3C
COOH
Lactate dehydrogenase
(乳酸脱氢酶)
Lactate
(乳酸)
D-Glucose + H2O + O2
H2O2 + dyereduced
Glucose oxidase
Peroxidase
C
COOH
Pyruvate
(丙酮酸)
D-gluconic acid + H2O2
H2O + dyeoxidized
30
P.335
6. Separation, purification and
storage of enzymes
I. Introduction
1) Separation & purification
(分离与纯化)
2) Storage (保存)
31
1). Separation and purification
I. Introduction
32
2). Storage of enzymes
Stored at high concentration, as lyophilized
powders, or in a concentrated (NH4)2SO4 solution
 Some proteases may go through autolysis
during storage.
 Some enzymes are easier to subject to
denaturation at low concentrations.
 Stored at low temperatures
 Be careful: freeze-thaw cycles would inactivate
enzymes.
 Don’t store protein samples in glassware.

I. Introduction
33
II. Enzyme Kinetics
(酶促反应动力学)
1. Review of chemical kinetics (化学动力学)
2. V-[S] relationship for an enzymatic reaction
(酶反应速度与底物浓度的关系)
3. Enzyme inhibition (酶的抑制作用)
4. Factors affecting enzyme activity (影响酶活
性的因素)
Enzyme kinetics
Applications
Reaction rate
Binding affinities for substrates and inhibitors
Mechanism
To achieve maximum catalytic efficiency
To understand metabolism
To control & manipulate metabolic courses
(e.g. pharmacology)
34
1. Review of chemical kinetics
P.351
(化学动力学)
SP
II. Enzyme kinetics
1)
2)
3)
4)
5)
6)
7)
d[P]
v =  d[S] =
= k[S]n
dt
dt
Reaction rate (反应速度)
v
Rate constant (速度常数)
k
Reaction’s order (反应级数)
n
Rate equation
v - [S] relationship
Half life(半衰期)
t1/2 (when [S] = ½[S]o, t = t1/2)
Activation energy (活化能)
Ea
Arrhenius equation
k = Ae-Ea/RT
35
Two general ways to lower activation energies
and hence to increase reaction rates

Raising the reaction temperature
-- To increase the average energy of reactants
-- Reaction rates are usually doubled by a 10oC rise in temperature

Adding catalysts
II. Enzyme kinetics
-- To lower the energy of the transition state
-- Regenerated after each reaction cycle  can be reused
-- No effect on overall free energy change  No change in direction
Transition state
Transition state
Ea(T1)Ea(T2)
S
Ea(uncat)
S
P
Raising the temperature
Ea(cat)
P
Adding a catalyst
36
P.352-355
Chemical kinetics (化学动力学)
Reaction type
(反应类型)
Zero-order reaction
(零级反应)
Rate
(速度)
[Substrate]
(底物浓度)
v=k
x = kt
Half life
(半衰期)
t1/2 =
a
2k
ln2
First-order reaction v = kc
c = coe-kt
t1/2 = k
(一级反应)
AP
1
b(a-x)
t
=
Second-order reaction v = kc1c2 ln a(b-x) = kt(a-b) 1/2 ka
(二级反应)
A+BP
a, b – initial concentrations of the reactants (初始浓度)
x – concentration that has gone through reaction after a
reaction period of t (反应t时已发生反应的物质浓度).
37
2. V-[S] relationship for an
enzymatic reaction
P.355
(酶反应速度与底物浓度的关系)
II. Enzyme kinetics
1)
2)
3)
4)
V-[S] plot (反应速度与底物浓度作图)
Michaelis-Menten equation (米氏方程)
Kinetic parameters (动力学参数)
Determination of Km, Vmax
38
1). V-[S] plot
P.355
Two models
V-[S] plot
ES complex model (中间复合物学说)
-- by Victor Henri (1903)
k E+P
E + S fast ES slow
Ks
Steady-state theory (稳态理论)
-- Briggs and Haldane (1925)
E+S
k1
k2
ES
k3
E+P
k4
Michaelis-Menten equation (1913):
Vmax [S]
V=
Km + [S]
39
II. Enzyme kinetics
40
P.356
Michaelis-Menten Equation: ES complex theory
(从中间复合物学说推导米氏方程)
Dissociation constant (解离常数)
Two-step reaction:
E+S
ES
Ks
k
ES (fast)
[E][S]
Ks =
[ES]
E + P (slow, rate-limiting step)
From (1)
[E]o[S]
[ES] =
Ks + [S]
From (2)
k[E]o[S]
V = k[ES] =
Ks + [S]
Vmax = k [E]o
Km = Ks
V=
=
([E]o[ES])[S]
[ES]
V = k [ES]
(1)
(2)
Vmax [S]
Km + [S]
41
Michaelis-Menten Equation: Steady-state theory
(从稳态理论推导米氏方程)
E+S
k1
k2
Rate of ES formation:
Rate of ES breakdown:
Rate of ES breakdown:
Rate of ES formation:
At steady state, V1 = V2 + V3
[E] = [E]o  [ES]
k2 + k3
Km =
k1
Vmax = k3 [E]o
ES
k3
E+P
d[ES]
dt = 0
k4
V1 = k1 [E][S]
V2 = k2 [ES]
V3 = k3 [ES] – Rate-limiting step
V4 = k4 [E][P] – Neglected
k1[E][S] = k2[ES] + k3[ES]
[E]o[S]
[ES] =
Km + [S]
V=
V = V3 =
k3 [E]o[S]
Km + [S]
Vmax [S]
Km + [S]
42
2). Michaelis-Menten equation (米氏方程)
P.358
v=
Zero-order reaction
Vmax[S]
Km + [S]
最大速率
Mixed-order reaction
When [S] ≫ Km,
v = Vmax
When [S] = Km,
v = ½ Vmax
When [S] ≪ Km,
Vmax
v=
[S]
Km
First-order reaction
米氏常数
Km is the substrate concentration at which v is one-half of Vmax.
43
3) Kinetic parameters (动力学参数)
P.359
E+S
k1
k2
ES
k3
E+P
V=
k4
Vmax [S]
Km + [S]
ES的解离常数
米氏常数
k2 + k3
k2
Km =
= Ks (When k3 ≪ k2) Affinity for the substrate
=
(酶对底物的亲和程度)
k1
k1
最大速率
Vmax = k3 [E]o = kcat [E]o
Turnover number
(转化数)
Specificity constant
(专一性常数)
Vmax
kcat =
[E]o
kcat/Km
kcat[E]o[S]
V=
Km + [S]
Catalytic efficiency
(酶的催化效率)
44
What can Km tell us and what can we do with the Km?
Km = [S] when v = ½ Vmax
k2 + k3
k2
Km =
= Ks (When k3 ≪ k2)
=
k1
k1
 At which [S], V = ½ Vmax
 Km can help to evaluate the reaction rate (估计反应速度)
 Km is a constant characteristic of the enzyme and the substrate
under specified conditions (是酶的特性常数，只与酶和底物的性质及反
应条件有关). e.g., an enzyme can have different Km’s for different substrates.
 Km may reflect the affinity of the enzyme for its substrate (反映酶和
底物结合的亲和程度)
 Km can help to evaluate the substrate specificity and to study the
active site of the enzyme (帮助评价底物专一性和研究酶的活性中心)
 Km can help to estimate the reaction direction and pathway (帮助推
断反应方向和途径)
45
Km values for some enzymes
Enzyme
Chymotrypsin
II. Enzyme kinetics
Substrate
Km (mM)
N-Benzoyltyrosinamide
2.5
Acetyl-L-tryptophanamide
5
N-Formyltyrosinamide
12
N-Acetyltyrosinamide
32
Glycyltyrosinamide
122
Pyruvate carboxylase
HCO31.0
Pyruvate
0.4
ATP
0.06
Hexokinase
Glucose
0.15
Fructose
1.5
Lactose
4 46
Vmax (最大反应速率)
v = k3 [ES]
v = Vmax
[S] » Km
Vmax = k3 [E]o
[ES] = [E]o
all enzyme molecules are saturated with substrate
E+S
E+S
k1
k2
k1
k2
k3
ES slow E + P
ES
k3
k4
k3 is rate-limiting, Vmax = k3 [E]o
k5
EP slow E + P
k5 is rate-limiting, Vmax = k5 [E]o
 It is necessary to define a more general rate constant, kcat, to describe
the limiting rate of any enzyme-catalyzed reaction at saturation.
Vmax = kcat [E]o
kcat is a first-order rate constant (s-1).
47
P.361
kcat, turnover number (转化数)
kcat 是一级反应速度常数 (s-1)
Vmax
kcat =
=
[E]o
=
=
d[P]
dt
[E]o
When all the enzyme
=
/sec molecules are saturated
Total [E]
with substrate
[P]
Moles of product being produced
/sec
Moles of enzyme
No. of substrate molecules being converted
kcat：体现酶
的催化效率
/sec
No. of enzyme molecules
The turnover number is equivalent to the number of substrate
molecules converted to product in a given unit of time on a single
enzyme molecule when the enzyme is saturated with the substrate.
kcat reflects the catalytic efficiency of an enzyme, and is a
measure of its maximal catalytic activity.
48
Values of kcat (turnover number) for some enzymes
II. Enzyme kinetics
Enzyme
Catalase (过氧化氢酶)
Carbonic anhydrase
Acetylcholinesterase
Penicillinase
Lactate dehydrogenase
Chymotrypsin
DNA polymerase I
Lysozyme (溶菌酶)
kcat (sec-1)
40,000,000
1,000,000
14,000
2,000
1,000
100
15
0.5
49
P.361
kcat/Km (specificity constant)
V=
Vmax [S]
Km + [S]
II. Enzyme kinetics
kcat [E]o[S]
=
Km + [S]
=
kcat
[E][S]
Km
(∵Vmax = kcat [E]o)
(Take [E]o as a variant)
 Apparent second-order rate constant (M-1s-1)
 Index of catalytic efficiency of an enzyme operating
at [S] substantially below saturation amounts
50
kcat vs. kcat/Km
kcat
kcat/Km
Turnover number
Specificity constant
Vmax = kcat [E]
v = (kcat/Km)[E][S]
Rate constant
First-order
(一级速度常数)
Second-order
(二级速度常数)
Represent
v when [S] >> Km: Vmax
v when [S] << Km
Unit
s-1
M-1s-1
V = kcat [E]
v = (kcat/Km)[E][S]
51
Km and kcat for some enzymes: Catalytic perfection
E+S
k1
k2
ES
k3
k4
E+P
kcat
k3k1
=
Km
k2 + k3

k3k1
 k1
k2
52
P.361
4) Determination of Km and Vmax
(作图法测定Km和Vmax值)
Lineweaver-Burk plot
1/v
Eadie-Hofstee plot
[S]/v
v
Slope = Km/Vmax
Hanes plot
Slope = 1/Vmax
Vmax
Slope = Km
1/Km
Km
1/Vmax
1/[S]
Km
1
1
1
=
+
v
Vmax [S]
Vmax
Double-reciprocal plot
(双倒数作图法)
v/[S]
v = Vmax  Km
[S]
v
[S]
[S]
v
=
[S]
Vmax
+
Km
Vmax
53
3. Enzyme inhibition (酶的抑制作用)
II. Enzyme kinetics
(1) Some terms related to enzyme inhibition
(2) Reversible inhibitors vs. irreversible
inhibitors (可逆抑制和不可逆抑制)
(3) Models of reversible enzyme inhibitions
(可逆抑制的三种类型)
(4) Application examples
54
(1) Some terms related to enzyme inhibition
Denaturation (变性作用) – Protein structure is changed
Inactivation (失活作用) – Loss of activity (the enzyme is denatured)
Inhibition (抑制作用) – A change in the chemical properties of some
functional groups in the enzyme  the loss or decrease in enzyme
activity (the enzyme is not denatured)
Inhibitors (抑制剂) -- selective
Denaturants (变性剂) – non-selective
Competitive inhibition
(竞争性抑制)
P.368
Reversible inhibition
(可逆抑制)
Inhibition
(抑制)
Irreversible inhibition
(不可逆抑制)
Noncompetitive inhibition
(非竞争性抑制)
Uncompetitive inhibition
(反竞争性抑制)
55
(2). Reversible inhibitors vs. irreversible inhibitors
P.369
(可逆抑制和不可逆抑制的鉴别)
 Physical methods
 Dialysis (透析)
 Gel filtration
 Ultrafiltration
(超滤)
 Kinetic method
No inhibitor
V
Irreversible inhibitor
(不可逆抑制剂)
-- Covalent interaction (共价结合)
-- Stable noncovalent association
(稳定的非共价结合)
-- Can’t be removed (不能除去)
Reversible inhibitor
(可逆抑制剂)
-- Noncovalent binding
(非共价结合)
-- Can be removed
[E]
56
Reversible inhibitor
Irreversible inhibitor
[I]
V
V
[I]
[E]
[E]
57
P.370
2). Models of Reversible Enzyme Inhibition
(可逆抑制的三种类型)
竞争性抑制
Vmax unchanged
Km 
非竞争性抑制
Vmax 
Km unchanged
反竞争性抑制
Vmax 
Km 
58
Competitive inhibition
(竞争性抑制)
Vmax unchanged
Km 
59
Noncompetitive inhibition
(非竞争性抑制)
Vmax 
Km unchanged
[I]
1
1
[I]
Km
1
=
+
)
(1 +
(1 +
)
v
V
max
Vmax
Ki [S]
Ki
1/Vo
[I]
-1/Km
1/[S]
60
Uncompetitive inhibition
(反竞争性抑制)
Vmax 
Km 
61
Kinetic effect on inhibited reaction
Type of inhibition
Km
Vmax
Vmax/Km
Competitive

unchanged

unchanged




unchanged
(竞争性抑制)
Noncompetitive
(非竞争性抑制)
Uncompetitive
(反竞争性抑制)
62
3) Application examples (应用举例)



Medical therapy – methanol poisoning (甲醇中毒的治疗)
Enzyme research – chymotrypsin (胰凝乳蛋白酶)
Rational drug design – penicillin (青霉素)
II. Enzyme kinetics
DIFP (Diisopropylfluorophosphate), nerve gas poison
Reaction of chymotrypsin with DIFP (irreversible inhibitor)
63
An example of drug design
based on enzyme inhibition
Penicillin
Peptidoglycan (肽聚糖)
– major component of the
cell wall of a bacterium
Muropeptide (胞壁肽)
– structural unit of
peptidoglycan
Glycopeptide
Penicillin
transpeptidase (糖肽转肽酶)
prevents the
– an enzyme to cross-link
synthesis of
cross-links
the peptidoglycan chains
during synthesis of
bacterial cell walls
Penicillin, an antibiotic
– a suicide substrate for the
Muropeptide
enzyme, covalently
(胞壁肽)
reacting with the Ser at the
14
enzyme’s active site
64
Inhibition of glutamine
amidotransferases for cancer treatment
Gln  Glu
H2N
CH
C
Inhibitors
OH
CH2
Gln
CH2
C
O
NH2
O
H2N
CH
C
OH
CH2
Glu
CH2
C
OH
O
65
P.378
4. Factors affecting enzyme activity
(影响酶活性的因素)
II. Enzyme kinetics
1) Enzyme concentration (酶浓度)
2) Substrate concentration (底物浓度) – Substrate inhibition
(底物抑制)
3) Product concentration (产物浓度) – Product inhibition
(产物抑制)
4) Temperature (温度) – Optimal temperature (最适温度)
5) pH – Optimal pH (最适pH)
6) Buffer type (缓冲溶液类型)
kcat[E]o[S]
Vo =
7) Ionic strength (离子强度)
Km + [S]
8) Activators (激活剂)
9) Inhibitors (抑制剂)
k = A e-Ea/RT
cat
66
Effect of pH on enzyme activity

Each enzyme has its specific optimal pH,
at which its catalytic activity is optimal.
(每一个酶有一个最适pH)





Effect of pH on the enzyme (对酶的影响)



Varying range: pH 1.5-9.7
Pepsin (胃蛋白酶): optimum pH ~ 2
Chymotrypsin (糜蛋白酶): optimum pH ~8
Alkaline phosphatase (碱性磷酸酶): optimum
pH ~ 9.8
Affecting the ionic state of the active site (影
响活性中心的电离状态)
Affecting the secondary and tertiary structure
(影响酶的二/三级结构)
Effect of pH on the substrate (对底物的
影响)
67
Effect of reaction temperature on enzyme activity
 Each enzyme has an optimal
reaction temperature.
 Effect of reaction
temperature
 T < opt. Temp.: T, V
Ground state of substrates 
 Ea 
Q10 = 2
 T > opt. Temp.: T, V
T  Thermal denaturation
 inactivation
 Factors affecting opt. Temp.




pH
Ionic strength
Buffer type
……
Effect of reaction temperature on activity
of alkaline phosphatase in DEA buffer
(0.1 M, pH 9.8) in the presence of
different inorganic salts (0.8 M)
*Yang Z, Liu X-J, Chen C, *Halling PJ.
68
Biochim. Biophys. Acta (2010)
P.384
III. Enzyme Mechanism
(酶的作用机制)
1. Active site (酶的活性部位)
2. Enzyme-substrate interactions (酶和底物之间
的相互作用)
3. Effects of specific catalytic groups on the
enzyme catalysis (一些活性功能团对酶催化
的影响)
4. Chymotrypsin (胰凝乳蛋白酶)
69
P.384
1. Active site (酶的活性中心)
III. Enzyme mechanism
1) Responsible for binding the substrate and catalytically
transforming it. (结合底物并催化其转化成产物)
2) Two parts: Binding site (结合点) and catalytic site (催化点)
3) Locates in a crevice on the surface of the enzyme molecule.
(位于酶分子表面的一个裂缝中)
4) Is a three-dimensional region with several amino acid
residues involved. (是一个三维实体)
5) Occupies only 1-2% of the total volume of the enzyme
molecule. (只占整个酶分子体积的1-2%)
Dihydrofolate reductase
NADP+
Tetrahydrofolate
70
P.388
2. Enzyme-substrate interactions
(酶和底物之间的相互作用)
III. Enzyme mechanism
1)
2)
3)
4)
Weak noncovalent interactions (非共价结合)
108-fold
Proximity effect (邻近效应) -- 104
activation
Orientation effect (定向效应) -- 104
Complementarity (互补效应)
Switching an intermolecular reaction to an intramolecular reaction
71
Transesterification
III. Enzyme mechanism
Proximity effect
Orientation effect
72
Complementary binding
between enzyme and substrate
(酶和底物之间的互补结合)
III. Enzyme mechanism
Hydrophobic/hydrophilic properties
(疏水/亲水性质)
Charge (电荷)
Size (分子大小)
Shape (分子形状)
73
Substrate specificity of 3 serine proteases
(丝氨酸蛋白酶的底物专一性)
P.408, Fig.10-35
O
H
N
CH C
R1
O
H
N
CH C
R2
H
N
Substrate binding sites:
 Chymotrypsin – a hydrophobic pocket
 Trypsin – Asp
 Elastase – a shadow binding pocket
弹性蛋白酶
R1 = Gly, Ala
胰凝乳蛋白酶
R1 = Phe, Trp, Tyr
胰蛋白酶
R1 = Lys, Arg
74
Models for E-S complementary binding
(酶和底物之间的互补性结合的两个模型)
“Lock and key” (锁钥理论) – Fischer (1894)
An enzyme is structurally complementary to its substrate, fitting together like a
“lock and key”
“Induced fit” (诱导契合理论) – Koshland (1958)
Interaction of a protein and its substrate involves changes in the conformation
of one or both, resulting in a complementarily nice fit.
Induced fit in hexokinase
D-glucose
The process is truly interactive – inducing a change in conformations of both enzyme and substrate. 75
Enzyme-Substrate Complementary Interaction
A change in the enzyme’s active site:
complementary to the substrate or to the transition state?
III. Enzyme mechanism
76
P.390
3. Effects of specific catalytic groups on
the enzyme catalysis
(一些活性功能团对酶催化的影响)
III. Enzyme mechanism
1) Acid-base catalysis (酸碱催化)
2) Covalent catalysis (共价催化)
3) Metal ion catalysis (金属离子催化)
77
Acid-Base Catalysis (酸碱催化)
III. Enzyme mechanism
Specific acid-base catalysis
(狭义的酸碱催化)
H+ (H3O+)
OH-
General acid-base catalysis
(广义的酸碱催化)
HA (proton donor)
B- (proton acceptor)
78
Amino acids in general acid-base catalysis
(可用于广义酸碱催化的氨基酸)
III. Enzyme mechanism
Proton transfers are the most common biochemical reactions.
79
Covalent catalysis (共价催化)
A-B + H2O
A-B
E:
A-E + B:
A-OH + HB
H2O
A-OH + E: + HB
III. Enzyme mechanism
Acid-base catalysis
His57
His57
Covalent catalysis
80
Metal ion catalysis
III. Enzyme mechanism
Role of the metal ion:
 To orient the substrate for reaction
(帮助底物分子定向)
 To stabilize charged reaction transition states
(稳定带电荷的反应过渡态)
 To mediate oxidation-reduction reactions
(参与/协调氧化还原反应)
81
R
R
HO
OH
k1
N
N
Cu2+
Cu2+
N
k-1
N
O
Catalytic cycle for
oxidation of o-diphenols
to o-quinones by
tyrosinase
O
K1 =
H
Emet
k-1
N
k1
N
O
N
Cu
2+
2+
Cu
N
O
Vmax =
R
H
EmetD
R
k2
k4
O
O
O
k2 + k4
O
N
N
+
Cu
R
k2 k4 [E]0
Cu
+
N
N
Km =
k2 K 3 + k 4 K 1
k 2 + k4
Edeoxy
K3 =
O
N
O
Cu
Cu
N
N
O
2+
O
EoxyD
2+
O2
k-3
k3
k-3
N
Cu
k3
R
N
N
O
2+
Cu
O
2+
N
N
Eoxy
HO
OH
*Yang Z., et al. (2009) J. Biochem. 145(3): 355–364. Importance of82
the ionic nature of ionic liquids in affecting enzyme performance.
P.405
4. Chymotrypsin (胰凝乳蛋白酶)
Serine protease
(丝氨酸蛋白酶)
Protein structure
(蛋白质结构)
Catalytic
mechanism
(催化机理)
Hydrolytic cleavage of peptide bonds
 Substrate specificity (R1 = Phe, Trp, Tyr)
 MW 25,000
 3 polypeptide chains (三条肽链)
 5 disulfide bonds (五条二硫键)

 Acyl-enzyme mechanism (酰基酶催化机制)
 Catalytic triad (催化三联体): Ser-His-Asp
 Transition-state stabilization (过渡态稳定化)
 Covalent and general acid-base catalysis (共价和酸碱催化)
 Two phases: acylation (酰基化) & deacylation (脱酰基化)
O
H
N
R1 = Phe, Trp, Tyr
H
C
R1
C
O
H
N
H
C
R2
C
H
N
83
Binding site
Structure of Chymotrypsin
Oxyanion hole
Carbonyl oxygen
Catalytic site
Disulfide bond
Peptide bond to be cleaved
84
Acyl-Enzyme Mechanism
Phase I: acylation (酰基化)
O-
O
R1
C
H
N
E-OH
R2
(酶)
R1
C
O
H
N
R2
R1
III. Enzyme mechanism
R2-NH2
O-E
Tetrahedral
intermediate
(E-S四面体中间物)
R1
C
OH
R1
E-OH
C
O-E
O-E
Acyl-enzyme
intermediate
(酰基酶中间物)
H2O
O-
O
C
OH
Tetrahedral
intermediate
(E-P四面体中间物)
Phase II: deacylation (脱酰基化)
85
86
Chymotrypsin Mechanism
Phase I: Acylation (酰基化)
Catalytic triad
(催化三联体)
Oxyanion hole
Acyl-enzyme intermediate
(酰基酶中间体)
(4)
Polypeptide substrate
Hydrophobic pocket
(3)
(1)
Tetrahedral intermediate
(E-S)
(2)
E-S complex
(酶-底物复合体)
87
Chymotrypsin Mechanism
Phase II: Deacylation (脱酰基化)
Tetrahedral intermediate
(E-P)
(6)
E-P complex
(5)
(7)
(4)
(酰基酶中间体)
Acyl-enzyme intermediate
88
P.344
IV. Introduction to enzyme engineering
(酶工程简介)
Enzyme research and application
Related areas/fields:
 Enzymology
(酶学)
 Molecular biology (分子生物学)
 Protein chemistry (蛋白质化学)
 Microbiology (微生物学)
 Immunology (免疫学)
 Chemistry (organic/inorganic/polymer…)
 Chemical engineering (化学工程)
 Material Science (材料科学)
 Clinical diagnosis (临床诊断)
89
Use of enzymes


Enzymes in intact organisms – direct use of microorganisms
Isolated enzymes

IV. Enzyme engineering



Modification (酶的修饰)
Chemical modification (化学修饰)
Site-directed mutagenesis (定点突变)
Directed evolution (定向进化)
Hybrid evolution (杂合进化)
Immobilization (酶的固定化)
Use in non-conventional media (酶用于非常规介质)
Organic solvents
Gases
Supercritical fluids (超临界流体)
Ionic liquids (离子液体)
Some special enzymes



Abzymes (抗体酶)
Ribozyme (核酶)
Artificial enzymes (人工酶)
90
第六届中国酶工程学术研讨会
会议内容
1. 国内外酶制剂生产及应用发展及前景
2. 酶研究的基础问题、热点及新生长点
3. 酶工程新技术、新工艺在新领域中的应用
4. 基因工程及蛋白质工程优良菌种及工程酶
5. 新酶、极端环境酶、抗体酶及核酶及其应用
6. 有机相酶促催化及有机化合物、药物、手性物生物合成
7. 环境监治与控制及洗涤剂用酶
8. 化工、轻工、饲料生产用酶的开发
9. 分析、临床诊断、医疗用酶的开发及生物传感器
10. 生物反应器与后处理工艺
91
Applications of enzyme engineering


IV. Enzyme engineering






Chemical industry
Food industry
Agriculture
Pharmaceutical industry
Clinical diagnostics and medical treatments
Biomedical engineering
Environmental protection
Exploitation of energy resources
92