酶 Enzymes
contents
• Introduction of enzymes
• Catalytic Mechanisms of enzymes
• Enzymatic reactionkinetics
Enzymes are biocatalysts with high
specificity and highly catalytic efficency
produced by living cells.
Besides proteins, ribozymes and
deoxyribozymes are also enzymes.
What is enzyme?
Ⅰ Introduction of enzymes
( 1 ) producing site : living cells
( 2 ) property : most——proteins
a few——RNA DNA
( 3 ) function : biochemical catalyzation
Structure and Function of Enzymes
Structure of enzymes
enzymes
Simple enzymes——proteins
apoenzyme
prosthetic group
coenzyme
Conjugated enzymes(holoenzyme)
cofactor
1.Composition of Enzyme Molecules
Holoenzyme = Apoenzyme + Cofactors
2. Active site of an enzyme
It is an area of enzyme;It is consist of a few amino acids residues;The residues are closed to each other in space;It combines substrate and catalyzes a reaction.
substrate Essential groups
outside of active site
Binding groups
catalytic group
active site
Active site of enzymes
Primary structure and spatial structure of chymotrypsin
3. Structure of enzyme
Three-dimensional structure Substrate binding
Ⅱ Properties and Catalytic Mechanisms of Enzymes
2. Highly Specificity of Enzymes
3. Activities of enzymes can be regulated
1. Highly Catalytic Activity of Enzymes
Properties of Enzymes
Catalytic Mechanisms of enzymes
Enzymes accelerate reactions by decreasing the activation energy.
For a biochemical reaction to proceed, the energy barrier needed to transform the substrate molecules into the transition state has to overcome. The transition state has the highest free energy in the energy pathway.
Formation of Enzyme-substrate Complex and Induced-fit Hypothesis
The combination of substrate and enzyme forces the substrate to become the transition state.
Energy diagram for catalyzed and uncatalyzed reactions .
Enzyme-substrate complex
Induced fit model
Induced fit model
Active sites in
the uninduced
enzyme
Binding of the first substrate (gold)
induces a conformational shift that
facilitates binding of the second
substrate (blue), with far lower
energy than otherwise required.
When catalysis
is complete, the
product is
released, and
the enzyme
returns to its
uninduced
state.
Hexokinase-D-glucose complexHexokinaseInduced fit
(1) Proximity and orientation effects
Catalytic mechanisms of enzymes
FIGURE 3-7 Proximity effects and orientation arrange
(2) Electrostatic effects
The active sites of enzymes are often hydrophobic.
Water is largedly excluded from the active site because of
the low dielectric constant. This increases the
electrostatic interactions between enzymes and
substrates and accelerate velocity of the reaction.
(3) Acid-base catalysis
Side chain groups in enzyme active sites, act as
proton donors or acceptors are termed general acids or
general bases.
The transition state is stabilized and the rate of
reaction is increased by adding or removing proton from
reactants. These chemical groups are imidazole group,
amino group, carboxyl group, and so on.
(4) Covalent catalysis
The attack of nucleophilic
or electrophilic group in the
enzyme active site upon the
substrate results in covalent
binding of substrate to the
enzyme as an intermediate in
the cause of catalysis.
Ser-OH
—CH2—S· ·
:H
—CH2—O· ·
:H
Cys-SH
—CH2—C=CH
HN N
CH
:His-imidazole
nucleophilic
Ⅲ Enzymatic reaction kinetics
ⅠⅠEffect of Effect of concentration of substratesconcentration of substrates on on enzyme reaction speedenzyme reaction speed
1.
2. sense and application of Km( 米氏常数 )
sense sense ::
(( 11 )) KmKm ,, that is the concentration of substrate that is the concentration of substrate
when reaction speed reach to ½ V when reaction speed reach to ½ V maxmax. .
(( 22 )) Km can present the affinity between enzyme and Km can present the affinity between enzyme and
substrate.substrate.
(( 33 )) Km is the mark constant.Km is the mark constant.
Application:Application:
(( 11 )) To be used to identify enzyme; To be used to identify enzyme;
(( 22 )) To estimate the best substrate of enzyme; To estimate the best substrate of enzyme;
(( 33 )) To calculate the concentration of substrate at the To calculate the concentration of substrate at the
fixed speed;fixed speed;
(( 44 )) To infer whether the enzyme is regulated by To infer whether the enzyme is regulated by
concentration of substrate;concentration of substrate;
(( 55 )) To estimate the trend of reaction.To estimate the trend of reaction.
3. Calculation metheds of Km3. Calculation metheds of Km
1.1. 双倒数作图双倒数作图
林贝氏方程林贝氏方程
V[S] Km
Vmax[S]
Vmax+=
1
-Km
ⅡEffect of pH on enzyme reaction speed
1. affect the combination of enzyme 1. affect the combination of enzyme and substrateand substrate
2. affect the conformation of enzyme 2. affect the conformation of enzyme moleculemolecule
动物体内多数酶的最适动物体内多数酶的最适 pHpH 接近中性 接近中性
Ⅲ Ⅲ Effect of Effect of temperature temperature on enzyme reaction speedon enzyme reaction speed
10 20 30 40 50 60 70 80 900
20
40
60
80
100
Temperature OC
Rel
ativ
e A
ctiv
ity
(%)
最适温度
Ⅳ Effect of concentration of enzyme on reation speed
activator:activator: the substance that could the substance that could
increase the activity of enzyme and increase the activity of enzyme and
elevate the enzyme reaction speed.elevate the enzyme reaction speed.
Ⅴ Effect of activator on enzyme reation speed
Inhibitor: Inhibitor:
the substance could decrease the the substance could decrease the
activity of enzyme.activity of enzyme.
Ⅵ Effect of inhibitor on enzyme reation speed
Types of inhibitingTypes of inhibiting
1. irreversible inhibition1. irreversible inhibition
specifity inhibitionspecifity inhibition
non-specifity inhibitionnon-specifity inhibition
2. reversible inhibition2. reversible inhibition
competitive inhibitioncompetitive inhibition
noncompetitive inhibitionnoncompetitive inhibition
uncompetitive inhibitionuncompetitive inhibition
non-specifity inhibitionnon-specifity inhibition
Cl
As
Cl
CH CHCl + E
SH
SHE
S
As
S
CH CHCl + 2HCl
1. irreversible inhibition1. irreversible inhibition
specifity inhibitionspecifity inhibition
R O
R' O
P
O
X
+ HO E
R O
R' O
P
O
O E
+ HX
the inhibitor was attached to enzyme with the inhibitor was attached to enzyme with
non covalent bond to decrease or lose the non covalent bond to decrease or lose the
activity of enzyme, but the inhibitor could be activity of enzyme, but the inhibitor could be
remove by dialysis.remove by dialysis.
2. reversible inhibition2. reversible inhibition
competitive inhibitioncompetitive inhibition
双倒数作图双倒数作图
Characteristic of competitive inhibitionCharacteristic of competitive inhibition
⑴ ⑴ 抑制剂往往是酶的抑制剂往往是酶的底物结构类似物底物结构类似物
⑵ ⑵ 抑制剂、底物与酶的抑制剂、底物与酶的结合部位相同结合部位相同—— —— 酶 酶
的活性中心的活性中心
⑶ ⑶ 抑制作用可以被抑制作用可以被高浓度的底物高浓度的底物减低以致消除减低以致消除
⑷ ⑷ (( 表观)表观) KmKm 值增大,值增大, VmVm 值不变值不变
noncompetitive inhibitionnoncompetitive inhibition
双倒数作图双倒数作图
⑴ ⑴ 非竞争性非竞争性抑制剂结构不一定与底物抑制剂结构不一定与底物的的结构类似结构类似;;⑵ ⑵ 抑制剂与酶的抑制剂与酶的活性中心外的位点活性中心外的位点结合;结合;⑶ ⑶ 底物浓度的改变对抑制程度无影响,底物浓度的改变对抑制程度无影响,抑制程度取抑制程度取决于抑制剂的浓度;决于抑制剂的浓度;
⑷ ⑷ 动力学参数:动力学参数: KmKm 值不变,值不变, (( 表观表观 )Vm)Vm 值降低值降低。 。
Characteristic of noncompetitive inhibitionCharacteristic of noncompetitive inhibition
uncompetitive inhibitionuncompetitive inhibition
双倒数作图双倒数作图
(( 11 ) 反竞争性抑制剂只与酶) 反竞争性抑制剂只与酶————底物的中间产底物的中间产
物结合,不与单独的酶结合。物结合,不与单独的酶结合。
(( 22 )动力学参数:)动力学参数: (( 表观表观 )Km)Km 、、 VmaxVmax 都变都变
小。小。
(( 33 )抑制程度与)抑制程度与 [S] [S] 和和 [ I ][ I ] 成正比。成正比。
Characteristic of uncompetitive inhibitionCharacteristic of uncompetitive inhibition
comparison of comparison of reversible inhibitionreversible inhibition