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Chapter Three
Enzymes
目录
Main contents
Introduction
1. Structure and Function of Enzymes
2. Nomenclature and Classification of
Enzymes
3. Properties and Catalytic Mechanisms of
Enzymes
4. Kinetics of Enzyme-catalyzed Reactions
5. Regulation of Enzymse Activity
6. Clinical Applications of Enzymes
Disease cases
Concepts
Questions
目录
Concept of Enzyme

Enzymes are the reaction catalysts of
biological systems with extraordinary
catalytic power and high degree of
specificity for their substrates, which are
produced by zoetic cells in organisms.

Category of Biocatalysts

Enzymes

Ribozymes ( Deoxyribozymes )
目录
The importance of enzymes to human
- Essential to every biochemical process
-Act in organized sequences to catalyze 100’s
of stepwise reactions by which
- Nutrient molecules are degraded
- Chemical energy conserved and transformed
- Macromolecules made from simple precursors
目录
History of Enzyme Research

BC 2000, there were records about ferment or made wine in
our country.

Before one century or so, Pasteur believed that the ferment
was the result of yeast activation in cells.

1877，Kuhne first brought forward the concept of “enzyme”

1897，Buchner brothers made the ferment success by using
the extracts of yeast from cells.

1926，Sumner successfully isolated the first enzyme----urease

1982，Cech first found that some special RNA molecules have
the activity to catalyze the RNA splicing, putting forward
“ribozyme” .

1995 ， Jack W.Szostak
deoxyribozyme
laboratory
first
reported
that
目录
Section One
The Molecular Structure
and Function of Enzyme
目录
1. Structure of Enzymes
1.1 Composition of Enzyme molecules
★ Simple enzyme
★ Conjugated enzyme
Protein part
Apoenzyme
holoenzyme
Small organic molecule
Cofactor Prosthetic group or coenzyme
Metal ion
目录
Classification of cofactors
according to how it combines to
apoenzyme
Coenzyme:
Combine with apoenzyme loosely, it could be
removed by ultrafiltration or dialysis
Prosthetic group:
Combine with apoenzyme tightly through
covalent bonds , it couldn’t be removed by
ultrafiltration or dialysis
目录
The actions of different components
in enzyme during a catalyst reaction
The specificity of a reaction decided
by apoenzyme
 The sort and character of a reaction
decided by cofactor

目录
目录
Some common coenzyme, their vitamin precursors
and deficiency disease
Coenzyme
Precursor
Deficiency disease
Coenzyme A
Pantothenic acid
Dermatitis
FAD, FMN
Riboflavin (vit B2)
Growth retardation
NAD+, NADP+
Niacin
Pellagra
Thiamine
Thiamine (vit B1)
Beriberi
Pyrophosphate
Tetrahydrofolate
Folic acid
Anemia
Deoxyadenosyl
Cobalamin (vit B12)
Pernicious anemia
Cobalamin
Co-substrate in the Vitamin C (ascorbic acid) Scurvy
hydroxylation
of proline
in collagen
Pyridoxal phosphate Pyridoxine (vit B6)
Dermatitis
目录
 Metalloenzyme
The combination of metal ion with enzyme
part is tight. It isn’t ease to lose the metal
ion during the isolation process.
 Metal-activated enzyme
The combination of metal ion with
enzyme part is not tight, but the metal
ion is needed for the activity of enzyme.
目录
 The actions of metal ions
Keep the conformation of enzyme stable；
Participate in the catalytic reaction, transfer
electrons；
Act as a bridge between enzyme and
substrate ；
Neutralize the negatively charged group,
decrease the electrostatic expel force
The actions of small organic molecules
Act as carrier to transfer electrons, protons or
other groups during the reaction process
目录
1.2 The various sorts of enzymes

Monomeric enzyme：only possess tertiary structure
Such as bovine ribonuclease



Oligomeric enzyme：consists of multiple homologous
or heterozygous subunits
Such as lactate dehydrogenase
Multienzyme system：multienzyme complex
aggregated by various functionally different enzymes
Such as pyruvate dehydrogenase complex
Multifunctional enzyme or tandem enzyme：an
enzyme with several different functions on the same
polypeptide chain because of the fusion event of
Such
as fatty acid synthase
genes during evolution
procedure
目录
2. Active Site of an Enzymes
Essential groups
That means the
chemical groups in
the polypeptide
chain related to the
activity of enzyme
molecule.
The primary and spatial structure of chymotrypsin
目录
Active center (or active site ) of enzyme
Definition----The active site of an enzyme is the
region of the enzyme that can binds the
substrate, to form an enzyme-substrate
complex, and transforms it into product.
The active site is a three-dimensional entity,
often a cleft or crevice on the surface of the
protein, in which the substrate is bound by
multiple weak interactions.
Two models : the lock-and-key model and the
induced-fit model.
目录

The essential groups inside the active site
Binding group
To bind with substrate
Catalytic group
To catalyze S to be P
The essential groups outside the active site
Be needed for keeping the active
conformation of enzyme, but presented
outside of the active site
目录
Essential group
outside active
site
substrate
Catalytic
group
Binding
group
Active site
目录
Active site of lysozyme
* Glu35 and Asp52 ---catalytic groups
* Trp62, 63, Asp101
and
Trp108---binding groups
* A~F are six Nacetylglucosamine
ring
Oligosaccharide chain of substrate
Active site of lysozyme
AA action sites of active site
目录
3. Structure and function of Enzymes
3.1 The Primary Structure of Enzymes
and its Function
The primary structure is the structural basis
for enzyme activity.
For example
Trypsin, chymotrypsin, elastase
Serine protease family, 25% homology, 4 –S-S目录
-Trypsin cleaves on the carboxyl side of
positively charged Lys or Arg residues
H2N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Lys-Ile-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr------
-Chymotrypsin cleaves on the carboxyl side of
bulky aromatic and hydrophobic amino acid
residues
H2N-Gly-Ile-Val-Glu-Trp-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Ser-Leu-Gln-Phe-Glu-Asn-------
-Elastase cleaves on the carboxyl side of
residues with small uncharged side-chains
H2N-Ile-Val-Glu-Ala-Cys-Cys-Thr-Ser-Ile-Cys-Trp-Ile-Gly-Gln-Phe-Glu-Asn------目录
Their differing specificities are determined by
the nature of the amino acid groups in their
substrate-binding sites which are
complementary to the substrates that they act
upon.
But all these three enzymes have a Ser195,
His57, Asp102, which are the main groups
involved in the catalytic mechanism.
目录
3.2 The Spatial Structure of Enzymes
and its Function
The catalytic activity of enzyme also depends
on its conformation such as trypsin etc
Trypsin binding site:
Gly216----Asp189----Gly226
Chymotrypsin binding site:
Gly216----Ser189----Gly226
To see fig 5-4
page 101
Elastase binding site:
Val216----Ser189----Val190
目录
Only intact enzyme possesses catalytic
activity
For an oligomeric enzyme, each single
subunit couldn’t exhibit activity.
-Trypsin
EC 3.4.21.4
- Chymotrypsin
EC 3.4.21.1
- Elastase
EC 3.4.21.36
目录
Section Two
The Nomenclature and
Classification of Enzyme
目录
1. Nomenclature of Enzyme
International Union of Biochemistry and
Molecular Biology (IUBMB)
Common naming method:
Each enzyme is named conventionally
with “ase” as suffix
Systematic naming method:
Substrate name + reaction type
EC + 4 digit number awarded by Enzyme
Commission (EC)
目录
-Enzymes are classified into six major
groups defined by the reaction that they
catalyze.
-Each enzyme has a unique four-digit
classification number according to the
classification criterion of enzyme brought
forward by International Enzyme
Commission (EC).
Samples
Urease EC 3.5.1.5
Hexokinase EC 2.7.1.1
目录
- Many named by adding suffix “ase”
to the name of their substrate or a
word describing their activity
Such as
lactate dehydrogenase
Pyruvate dehydrogenase
hexokinase
目录
2. Classification of Enzyme
(1) Oxidoreductases
(2) Transferases
(3) Hydrolases
(4) Lyases
(5) Isomerases
(6) Ligases, synthetases
OTHLIL
目录
International classification of enzymes
Class Name
1
2
3
4
5
6
Type of reaction catalyzed
Example
Oxidoreductases Transfer of electrons A-+B→A+B- alcohol
dehydrogenase
Transferases
Transfer of functional groups
Hexokinase
A-B + C →A + B-C
Hydrolases
Hydrolysis reactions
Trypsin
A-B + H2O →A-H + B-OH
Lyases
Cleavage of C-C, C-O, C-N
Pyruvate decarand other bonds, often forming
boxylase
a double bond
A-B →A=B + X-Y
Isomerases
Ligases
or synthases
XY
Transfer of groups within a molecule
A-B →A-B
XY Y X
Bond formation coupled
to ATP hydrolysis
A + B → A-B
Maleate
isomerase
Pyruvate
carboxylase
目录
Section Three
Properties and Catalytic
Mechanisms of Enzymes
目录
1. Properties of Enzyme Catalyzed
Reaction
The common characters of an enzyme and a
general catalyst :
* Before or after a reaction, enzymes are not
changed themselves；
* Enzymes do not alter the equilibrium position,
but do accelerate the attainment of the equilibrium
position by speeding up the forward and reverse
reactions.
* The reaction is thermodynamically permissive.
目录
The Characters of enzyme catalysis
(1) High effectively catalytic rate ( The
rate can be increased up to 108~1020fold).
(2) Highly specificity
(3) Reaction enzyme-catalyzed can be
regulated.
目录
1.1 Highly Catalytic Ability of Enzymes
(1) High effectively catalytic rate ( The
rate can be increased up to 108~1020fold).
(2) No high temperature is needed
(3) Can more effectively decrease
activation energy of reaction
the
目录
High effectively catalytic ability
Mechanisms
To decrease the activation energy of
reaction
* The number of active molecules ↑
* More molecules easily become active
molecules
目录
Gibbs free energy of activation (ΔG‡ )
Non-enzyme
-catalyzed
reaction
free energy
S=substrate
P=product
Transition state
ΔG‡
General catalyzed reaction
Enzyme-catalyzed
reaction
S
ΔG (total free
energy change)
P
Progress of reaction
The energy changes taking place during the
course of a biochemical reaction
目录
Activation energy and transition state
Energy barrier ----In all reactions there
is an energy barrier that need to
transform the substrate molecules into
the transition state.
Transition state----an unstable chemical
form part-way between the substrates
and the products
目录
Free energy change
The difference in energy level between
the substrates and products is termed
the change in Gibbs free energy (ΔG ).
A negative ΔG indicates that the
reaction is thermodynamically favorable
in the direction indicated, whereas a
positive ΔG indicates that the reaction is
not thermodynamically favorable and
requires an input of energy to proceed
in the direction indicated.
目录
An energetically unfavorable reaction
is often driven by linking it to an
energetically favorable reaction, such
as the hydrolysis of ATP.
Gibbs free energy of activation (ΔG‡ )
is equal to the difference in free energy
between the transition state and the
substrate.
目录
1.2 Highly Specificity of Enzymes
Definition----It is termed as specificity of
enzyme that an enzyme only can catalyze
one kind of substrate to react and to form
one kind of product.
Why?
Because there is active site of enzyme
which related to some amino acid
residues in peptide chain.
目录
The specificity of enzyme can be classified in
light of their substrates and actions:



Absolute specificity：Only catalyze one
special kind of substrate and produce one
kind of product
Relative specificity：Can catalyze one sort
of compounds or chemical bonds
Stereo specificity：Only act on a single
stereoisomer of the substrate
For example: LDH
目录
1.3 Activities of Enzymes Can Be
Regulated
 To regulate the amount of enzyme in
organism
For example: synthesis or degradation of
enzymes
 To regulate the catalytic effectiveness of
enzyme
For example: allosteric regulation,
covalent modification to enzymes
目录
2. Catalytic Mechanism of Enzymes
2.1 formation of ES Complex and Inducedfit hypothesis
Enzyme-substrate complex
E+S
ES
E+P
*induced-fit hypothesis
When an enzyme approaches to its substrate
mutually, the structure of enzyme could be changed
and adapted each other, then combines with the
substrate. This process is termed induced-fit
hypothesis.
目录
Induced fit hypothesis
E itself undergoes a change in conformation upon
binding S
- Permits formation of additional weak binding interactions in transition state
- Brings specific functional groups into proper position for catalysis
目录
The flash of induced-fit hypothesis
目录
The induced-fit of carboxypeptidase
substrate
目录
2.2 Abzymes
In 1948 Linus Pauling proposed that
compounds resembling the transition state of
a catalyzed reaction should be very effective
inhibitors of enzymes. These mimics are called
transition state analogs. The transition state
analogs use the hapten to generate antibodies
with catalytic activity. These antibodies are
called abzymes which can be used as new
enzymes and drugs.
目录
2.3 Several Factors Contributing to Enzyme
Catalysis
(1) Proximity and orientation effects
(2) Electrostatic effects
(3) Acid-base catalysis
(4) Covalent catalysis
目录
Section Four
Kinetics of EnzymeCatalyzed Reactions
目录

The concept of kinetics of enzymecatalyzed reactions
It is a research about the factors to influence
the velocity of enzyme-catalyzed reactions.

The factors including:
Conc. of enzyme, Conc. of substrate,
pH, temperature, inhibitors, activators, etc.
※ When one factor is studied, other factors
should be invariableness.
目录
V0
Amount of product
formed (μ mol )
V0
Time (min)
The relationship between
product formation and time
for an enzyme-catalyzed
reaction
[S]
The relationship between
substrate conc [S] and initial
reaction velocity (V0)
目录
1. The Effect of Substrate on the
Velocity of enzyme-catalyzed Reactions
1.1 Research Preconditions
I.
Only single substrate, single product
II.
The velocity of enzyme-catalyzed reaction
under certain conditions
III.
To measure the initial velocity when the
change of [S] is less than 5%
目录
What is velocity of enzyme?
Enzyme velocity are normally reported as values
at time zero ( symbol V0; μmol min-1)
In fact, V0 means the rate of an enzymecatalyzed reaction at beginning of reaction.
Why V0 should be used to represent an enzyme
activity?
1) The substrate conc is highest.
2) No feedback inhibition by product.
3) Enzyme activity is in best state.
目录
Enzyme velocity
Enzyme activity is commonly expressed by the
initial rate ( V0) of the reaction being catalyzed.
The units of V0 are μmol min-1
Two standard units of enzymes activity unit
(U) or the katal (Kat)
1 μmol min-1 = 1U = 16.67 nanokat.
Total activity refers to the total units of
enzyme in a sample
Specific activity is the number of units per
milligram of protein ( units mg-1)
目录
1.2 Rectangular Hyperbola Plot of Initial
Velocity Versus Substrate Concentration
-At low [S], V0 will increase with the increase
of [S]
-At higher [S], enzyme becomes saturated,
there would no increase of V0 even with the
increase of [S].
-A graph of V0 against [S] will give a
hyperbolic curve.
目录
Rectangular Hyperbola Curve
V0
[S]
The relationship between substrate conc
[S] and initial reaction velocity (V0)
目录
V
Vmax
[S]
-At low [S], V will increase at directly ratio with
the increase of [S]. It is the first order reaction
目录
V
Vmax
[S]
With the increase of [S], the velocity
continues increasing, but not at directly ratio.
It is a mixed-order reaction.
目录
V
Vmax
[S]
-At higher [S], enzyme becomes saturated, there
would no increase of V even with the increase of
[S]. Here, the velocity has reached its most
velocity. It is a zero-order reaction.
目录
1.3 Formulation of the Michaelis-Menten
Equation
The model of enzyme-catalyzed reaction —
—intermediate product hypothesis
E+S
k1
k2
ES
k3
E+P
intermediate product: ES
目录
※1913, Michaelis and Menten
Michaelis-Menten Equation
Ｖ
＝
Vmax[S]
Km + [S]
[S]： concentration of substrate
V：
velocity of reaction at different [S]
Vmax：maximum velocity
Km： Michaelis constant
目录
The Michaelis-Menten Equation was deduced in
light of two hypothesises:
The formation of ES is a quickly equilibrium
reaction, but ES decomposed to be product is
slowly, the speed of reaction dependents on the
slow reaction, namely V＝k3[ES].
(1)
The total [S] is much higher than [E], therefore, the
[S] could be considered no change at the initial
stage of reaction, [S]＝[St].
目录
The deductive process

In a steady state, the velocity of formation of ES is
equal to that of degradation, so, [ES] is equilibrium.
K1 ([Et]－[ES]) [S]＝K2 [ES] + K3 [ES]
([Et]－[ES])[S]
Coordinate that：
[ES]
Order：
K2+K3
＝ Km
＝
K2+K3
(2)
K1
Michaelis-Menten
Constant
K1
then(2)changed to: ([Et]－[ES]) [S] ＝Km [ES]
目录
Coordinate that：
[Et][S]
[ES]＝───
Km + [S]
(3)
Take (3) instead of (1), then,
V＝
K3[Et][S]
Km + [S]
(4)
V＝k3[ES]
(1)
目录
At very high [S], the active sites would be
saturated completely, so [Et]＝[ES], V would
reach the maximum value,
Vmax＝K3[ES]＝K3[Et]
(5)
Substituting (5) into (4), Michaelis-Menten
Equation is yielded:
Vmax[S]
V＝────
Km + [S]
目录
How to calculate Km value
V
When V is equal to half of Vm, it can be
deduced:
Vmax
2
Vmax
Vmax[S]
＝
Km + [S]
Vmax/2
Km＝[S]
Km
[S]
∴Km value is equal to the [S] at ½ of Vm ,its
unit is mol/L。
目录
1.4 The Significance of Km and Vm
Km value
① Km value is equal to the [S] at ½ of Vm ,its
unit is mol/L.
② Significances：
a) Km is one of characteristic constants of an
enzyme；
b) Km can be used to represent the affinity of
enzyme to its substrate; the larger, the less
affinity
c) There are different Km values for an enzyme
to different substrates
目录
Vmax
Definition：Vm is the velocity of enzymecatalytic reaction when enzyme is completely
saturated, directly proportional to [E].
Significance：Vmax=K3 [E]
If total [E] has been known, the turnover
number of an enzyme or kinetic constant K3
can be calculated in light of Vm.
目录
Turnover number of an enzyme
Definition —
The number of substrate molecules are
transferred into product in each unit of time
when the enzyme molecules are fully saturated
by substrates
Significance ----It can be used to compare the
catalytic ability of each unit of enzyme.
Generally, it is about 1~104 /sec for the turnover
number of most enzymes.
目录
1.5 Measurement of Km and Vm Values
Double Reciprocal Plot , or Lineweaver- Burk
Plot
V=
Vmax[S]
1/V
Km+[S]
By taking the reciprocal of
both sides of the MichaelisMenten equation
Km
1/V=
+ 1/Vmax
1/[S]
Vmax
-1/Km
1/Vm
1/[S]
Lineweaver- Burk Plot equation
目录
Double reciprocal plot（Lineweaver—Burk plot ）
Slope = Km/Vmax
1
V
1 Km 1
1
=
+
V Vm [ S ] Vm
Intercept
Plotting
1
[S]
1
V
against
Intercept
1
Vm
1
Km
1
[S]
Resulting in a double reciprocal
or Lineweaver-Burk plot
目录
2. The Effect of Enzyme on the Velocity
of enzyme-catalyzed Reactions


V
When [S] ＞ ＞ [E] ，
enzymes
could
be
saturated by substrates,
the velocity of enzymecatalytic
reaction
is
directly proportional to
the [E] .
The equation should be： 0
V = K3 [E]
[E]
When [S]>>[E]，Vmax = k3 [E]
目录
3. The Effect of Temperature on the
Velocity of enzyme-catalyzed Reactions
Optimum temperature
2.0
Temperature affects the rate 酶
of enzyme-catalyzed
活
1.5
reactions in two ways.
性
A rise in temperature
increases the thermal energy
of the substrate molecules
(at less than 40 ℃).
1.0
0.5
At higher temperature( >50℃),
0 10 20 30 40 50 60
enzymes are more easily
T ºC
denatured.
Amylase activity versus T
目录
The overall effect of a rise in temperature on
the reaction rate of the enzyme is a balance
between these two opposing effects.
The optimum Temperature
The temperature of reaction at point which
enzyme possesses maximal efficiency is
termed the optimum temperature for this
enzyme.
目录
4. The Effect of pH on the Velocity of
enzyme-catalyzed Reactions
Acetylcholine
esterase
amylase
pepsin
Enzyme activity
Optimum pH：
The pH at which
an enzyme has
maximal activity
in solution.
0
2
4
6
8
The effect of pH on V
10
pH
目录
5. The Effect of Inhibitors on the
Velocity of enzyme-catalyzed Reactions

Inhibitors of enzymes
The substances which can make the activity
of enzyme decreasing but not cause the enzyme
denaturation can be called the inhibitor of
enzymes
 Different from the denaturation of enzymes
• Inhibitor has itself selection for enzymes
• The factors causing enzyme denaturation have no
selection for enzymes
目录
Enzyme inhibition
The activities of an enzyme are easily affected
by many types of molecule which exist in
organisms. Some of them can decrease the
activities of enzymes, but others improve the
activities of enzymes.
Inhibitor----Any molecule which acts directly on
an enzyme to lower its catalytic rate is called an
inhibitor.
Source----normal cellular metabolites, drugs,
toxins (foreign substances )
目录
 The category of inhibition
Irreversible inhibition
Reversible inhibition：
Competitive inhibition
Non-competitive inhibition
Uncompetitive inhibition
目录
5.1 Irreversible Enzyme Inhibition
* Concept
Irreversible
inhibitors
usually
bind
covalently to the enzyme, often active groups ,
and make the enzyme lose its activity.
For example
Organophosphorus compound hydroxy enzyme
Detoxification --- PAM ( pyridine aldoxime
methyliodide)
Heavy metal ions or As (arsenic)  sulfhydryl
enzyme
Detoxification --- BAL (二巯基丙醇)
•
目录
R
O
O
+
P
R'
O
R
HO E
R'
Hydroxy
enzyme
Cl
CHCl
CH2
E
As
CH
S
Inactive E
CHCl
+
acid
CH
CHCl
+ 2HCl
Inactive E
SH
CH
SH
CH2
OH
BAL
E
S
Sulfhydryl
enzyme
S
As
E
SH
Lewisite
O
HX
S
+ E
Cl
O
Inactive E
SH
CH
+
P
X
Organophosphorus
compound
As
O
O
CH2
SH
S
As CH
+
E
acid
SH
Sulfhydryl
enzyme
CH
S
CH2
OH
CHCl
BAL binding with
arsenic
目录
5.2 Reversible Enzyme Inhibition
* Concept

Reversible----Inhibitors usually bind to
enzyme uncovalently. Enzyme inhibition
can be overcame by removing the
inhibitors from the enzyme, such as by
dialysis, ultrafiltration
* Category
a. Competitive inhibition
b. Noncompetitive inhibition
c. Uncompetitive inhibition
目录
(1) Competitive Inhibition
Definition
If a inhibitor could bind to the active site of
enzyme competitively instead of substrate due to that
the structure of inhibitor is similar to substrate’s, the
activity of the enzyme would be inhibited and
decreased. The inhibition is termed
competitive
inhibition.
Reaction model
E+S
ES
E+P
+
I
EI
目录
Competitive inhibition
Characteristics:
1) Inhibitor structure is similar to the
enzyme’s
2) Inhibitor and substrate can
competitively bind to the active site on
an enzyme.
3) At high [S], the inhibition can be
overcome.
4) Vmax keep unchanged but Km increase.
目录
Competitive inhibition
E
+
S
+
E
I
+
ES
E
P
EI
目录
Competitive inhibition
Km
[I] 1
1
1

(1  )

V Vmax
Ki [S] Vmax
Vmax [S]
V
[I]
K m (1 ) [S]
Ki
1/V
Inhibitors↑
Vmax keep unchanged
but Km increase.
No inhibitors
Km ↑, Vmax－
1/[S]
目录
* For example 1
* For example 2
Malonate and succinate can competitively bind
to succinate dehydrogenase
Succinate
Succinate dehydrogenase
FAD
Fumarate
FADH2
COOH
COOH
CH2
CH2
COOH
CH2
COOH
Malonate
丙二酸
目录
(2) Noncompetitive Inhibition
Reaction model
E+S
+
I
ES
+
I
EI+S
EIS
E+P
+S
E
－S
+
ES
E
P
+S
EI
－S
ESI
目录
Noncompetitive inhibition
Characteristics:
-A noncompetitive inhibitor binds
reversibly at a site other than the active
site and leads to a decrease in catalytic
activity.
-An enzyme can bind the inhibitor or
substrate or both.
-The effects of a noncompetitive inhibitor
cannot be overcome by increasing [S]
-Resulting in Vmax ↓, Km keep the same.
目录
Noncompetitive inhibition
Km
[I] 1
1
[I]
1

(1  )

(1  )
V Vmax
Ki [S] Vmax
Ki
1/V
Km keep unchanged
but Vmax decrease.
Km －, Vmax ↓
Inhibitor ↑
No inhibitor
1/[S]
目录
(3) Uncompetitive Inhibition
Reaction model
E+S
ES E+P
+
I
ESI
+
E
+
S
ES
E
P
ESI
目录
Uncompetitive Inhibition
Characteristics:
-A uncompetitive inhibitor only binds
reversibly ES complex, and leads to a
decrease in catalytic activity.
-The inhibition depends on the conc. of [S]
and [I].
-The effects of an uncompetitive inhibitor
cannot be overcome by increasing [S].
-Resulting in Vmax ↓, Km ↓.
目录
Uncompetitive Inhibition
1
1  Km
 1 (1+ [I] )
Ki
Vmax
V Vmax [S]
●
inhibitors↑
1/V
Km decrease and Vmax
decrease too.
No inhibitor
Vmax ↓, Km ↓
1/[S]
目录
Comparison of inhibition by different
reversible inhibitors
Properties
No
inhibitors
Component bind with I
Competitive
inhibitors
E
Noncompetitive
inhibitors
E, ES
Uncompetitive
inhibitors
ES
Apparent Km
Km
↑
unchanged
↓
Vmax
Vmax
unchanged
↓
↓
Intercept at X axis
-1/Km
↑
unchanged
↓
Intercept at Y axis
1/Vmax
unchanged
↑
Slop
Km/Vmax
↑
Lineweaver-burk plot
↑
↑
unchanged
目录
6. Activators of Enzymes

Activator
Any substance which can cause enzyme
activity increase can be termed as activator
of enzyme.
• essential activator
• non-essential activator
Generally, many kinds of metal ions are activators for
some enzymes, such as Mg2+, K+, and Mn2+
Magnesium, Mg2+; Potassium, K+, Manganese, Mn2+
目录
Section Five
Regulation of Enzyme
Activity
目录
The object to be regulated:
Key enzymes associated with the
committed steps
The manners of regulation
Regulation to the enzyme activity
(rapid regulation）
Regulation to the amount of enzyme
( slow regulation）
目录
1. Allosteric Regulation
Definition
The regulation of enzyme activity occurs
when some metabolites combine reversibly
to an allosteric site spatially remote from the
catalytic site of an enzyme and change the
conformation of the enzyme ,resulting in the
change of enzyme activity. This regulation is
termed allosteric regulation.
目录
• allosteric enzyme
•allosteric site
•
Allosteric activator
allosteric effectors
Allosteric inhibitor
目录
1.1 The Mechanism of Allosteric Regulations
The conformational change of an allosteric
enzyme can cause itself activity change,
increasing or decreasing, depending on the
effectors.
An alloseric activator increase the rate of
enzyme activity, while an allosteric inhibitor
decreases the activity of the enzyme.
Example:
Aspartate transcarbamoylase ( ATCase )
Its structure: 6 catalytic subunits + 6
regulatory subunits
目录
CO2 + glutamine + ATP
CPS II
CPS II----carbamoyl
phosphate synthetase II
COOO
CH2
H3N+ CH COO-
Activation
NH2
C
Carbamoyl phosphate
OPO32ATCase
H2PO4N-Carbamoylaspartate
O
ATCase----Aspartate
transcarbamoylase
NH2
C
N
H
COOCH2
CH COO-
Formation of Ncarbamoylaspartat
e by ATCase is the
committed step in
pyrimidine
biosynthesis and a
key control point
Inhibition
CTP
目录
+ ATP
V0
hyperbolic-shaped curve
No allosteric
effectors
- CTP
0
10
sigmoidal-shaped curve
20
30
Aspartate (mM)
Plot of initial reaction velocity (V0) against substrate
concentration for the allosteric enzyme ATCase
目录
1.2 General Properties of Allosteric Enzymes
Key points:
1) An allosteric enzyme is regulated by its effectors
(activator or inhibitor).
2) Allosteric effectors bind noncovalently to the
enzyme they regulate
3) Allosteric enzymes are often multi-subunit proteins.
Allosteric enzymes have often more than one active
site.
4) A plot of V0 against [S] for an allosteric enzyme
gives a sigmoidal-shaped curve.
5) The binding of one of subunits of an allosteric
enzyme with an effector will induce a conformational
change
目录
2. Covalent Modification
Definition---Reversible covalent modification is the
making and breaking of a covalent bond
between a nonprotein group and an enzyme
molecule.
Forms
of Covalent Modification
Phosphorylation / dephosphorylation
adenylylation/deadenylylation
methylation/demethylation
－SH / －S－S , etc
目录
Covalent Modification
Pi
Protein
phosphatase
H2 O
Protein-OH
O-
ATP
Protein kinase
Protein-O-P=O
O-
ADP
The reversible phosphorylation and
dephosphorylation of an enzyme
目录
Covalent Modification
Key points:
1) Change of a covalent bond
2) The most common is the addition and
removal of a phosphate group,
phosphorylation or dephosphorylation
3) Enzymes----protein kinases or phosphatases
4) The activity of an enzyme after the
modification can increase or decrease
5) The modification is a rapid, reversible and
effective process
目录
3. Zymogen
Concept---Several enzymes are synthesized as larger
inactive precursor forms called proenzymes or
zymogens.
That process involved irreversible hydrolysis of
one or more peptide bonds on a zymogen is
termed the activation of zymogen.
Examples:
Trypsin, chymotrypsin and elastase in the
pancreas
目录
trypsinogen
+
+
enteropeptidase
trypsin
Chymotrypsinogen
+
Chymotrypsin
+
proelastase
elastase
The central role of trypsin in activating the
pancreatic zymogens
目录
Blood clotting cascade
Another example of the occurrence of
inactive zymogens is found in the enzymes
involved in the blood clotting cascade.
The whole process of blood clotting is
brought about by series of zymogen
activations.
目录
enteropeptidase
trypsin
ValAspAspAspAspLys Ile Val Gly
His
46
S
Ser
18
3
S
S
S
Active site
Val AspAspAspAspLys
Val
Ile GlyHie
Ser
S
S
S
S
The process of trypsinogen activation
目录
4. Isoenzymes
Definition
Multiple forms of an enzyme that can
catalyze the same reaction but differ from
each other in their amino acid sequences,
substrate affinity, Vm, and /or regulatory
properties are called isoenzymes (or
isozymes )
目录
For example
Lactate dehydrogenase, LDH1～ LDH5)
H H
H H
H H
H M
H H
M M
H M
M M
M M
M M
LDH1
(H4)
LDH2
(H3M)
LDH3
(H2M2)
LDH4
(HM3)
LDH5
(M4)
Isoenzymes of LDH
目录
Myocardium case
Enzyme activity
Significances
It can be used in
diagnostic
differentiation of
the liver diseases
and myocardium.
Normal case
Liver disease
1
2
3
4
5
LDH isoenzyme electrophoresis map
目录
5. Genetic Control
-The amount of enzyme present is a balance
between the rates of its synthesis and
degradation.
-The level of induction or repression of the
gene encoding the enzyme, and the rate of
degradation of its mRNA, will alter the rate of
synthesis of the enzyme protein.
-Once the enzyme protein has been
synthesized, the rate of its breakdown (halflife ) can also be altered as a means of
regulating enzyme activity.
目录
Section Six
Clinical Applications of
Enzymes
目录
1. Enzymes and Pathogenesis
- defects = basis of genetic disorders
(eg. enzyme activity missing)
- basis of some cancers (eg. growth
controlling enzyme permanently “on”)
目录
Enzymes defects induce molecular diseases
Diseases
Enzymes defects
Albinism
Tyrosinase
Favism
Lesch-nyham
syndrome
Glucose-6-phosphate
dehydrogenase
Hypoxanthine guanine
phosphoribosyltransferase
Homocystinuria
Cystathionine synthase
Phenylketonuria
Phenylalanine hydroxylase
目录
Phenylalanine hydroxylase
Individuals lacking this enzyme
suffer from phenylketonuria,
PKU, an inborn error of
metabolism
目录
Lesch-Nyhan syndrome (LNS) is a rare
inherited disease that disrupts the metabolism
of the raw material of genes.
These raw materials are called purines, and
they are an essential part of DNA and RNA.
The body can either make purines (de novo
synthesis) or recycle them (the resalvage
pathway). Many enzymes are involved in these
pathways. When one of these enzymes is
missing, a wide range of problems can occur.
目录
In LNS, there is a mutation in the HPRT1 gene
located on the X chromosome. The product of
the normal gene is the enzyme hypoxanthineguanine phosphoribosyltransferase, which
speeds up the recycling of purines from
broken down DNA and RNA. Many different
types of mutations affect this gene, and the
result is a very low level of the enzyme.
目录
The molecular basis of glycogen
storage disease type 1a: structure and
function analysis of mutations in
glucose-6-phosphatase.
1: J Biol Chem 2002 Feb
15;277(7):5047-53Related Articles,
Links
目录
1: Blood 2003 Jan 1;101(1):345-7Related
Articles, Links
HK Utrecht: missense mutation in the active
site of human hexokinase associated with
hexokinase deficiency and severe
nonspherocytic hemolytic anemia.
van Wijk R, Rijksen G, Huizinga EG,
Nieuwenhuis HK, van Solinge WW.
Department of Clinical Chemistry and the
Department of Hematology, University Medical
Center Utrecht, The Netherlands.
目录
PABA, p-amino- + Glu + dihydropterin
benzoic acid
1
sulfanilamide inhibiting
dihydrofolic acid
2
1-dihydrofolate synthetase
Tetrahydrofolic acid
2-dihydrofolate reductase
Go to 87
目录
2. Enzymes and Diagnosis of the
Diseases
2.1 Blood Plasma Enzymes and
Diagnosis of Diseases
Some enzymes presenting in high concentration in
blood plasma have important functions such as blood
coagulation ( thrombin ), fibrin dissolution ( plasmin )
and processing of chylomicrons (lipoprotein lipase )
Some enzymes presenting in trace amount in blood
plasma generally come from other tissues, which is
called intracellular enzymes. The change of the
amount of them in plasma would give some
information about the tissues normal or nonnormal.
目录
The blood plasma enzymes for disease diagnosis
Enzymes
Source
Diagnosis for the disease
Amylse
Saliva pancreas, ovary
Pancreas disease
Alkaline
phosphatase
Liver, bone, kidney, mucosa
of intestine
Bone, liver disease
Acid
phosphatase
GPT
Red blood cells, prostate
gland
Liver, heart, skeletal muscle
Liver, heart, skeletal muscle,
GOT
kidney, red blood cells
Skeletal muscle, brain, heart,
Creatine kinase smooth muscle
Liver, heart, skeletal muscle,
LDH
red cells, platelet, lymphoid
node
Choline esterase Liver
Caner of prostate gland,
bone disease
Liver disease
Myocardial infarction,
liver muscle disease
Myocardial infarction,
muscle disease
Myocardial infarction,
hemolysis, liver disease
Organophosphorus
poison, liver disease
目录
2.2 Immobilized Enyzme and
diagnosis of Diseases
2.3 Enzyme-linked Immunoassays
and Diagnosis of Diseases
2.4 Isoenzyme and Diagnosis of
Diseases
2.5 Insoluble enzymes and
Diagnosis of Diseases
目录
3. Enzymes and Therapy of Diseases
Some enzymes can be used as therapeutic agents.
Strepotokinase, for the clearing of blood clots
Asparaginase therapy, for the leucocythemia
Intravenous administration of asparaginase could
reduce the host’ plasma level of asparagine, which
causes tumor regression.
目录
Disease cases
1. Hepatitis (肝炎)
2. Adenosine deaminase, ADA
(腺苷脱氨酶缺乏)
3. Macrocytic anemia ( 巨红细胞贫血)
目录
Concepts
1 Biocatalysts
2 Enzymes, apoenzyme, coenzyme
3 Active Site of Enzyme
4 Allosteric regulation
5 chemical modification
6 Isoenzymes
7 zymogen
8 Km, Vm
9
reversible
inhibition,
irreversible
inhibition
10 competitive inhibition
目录
Questions
1 What is the substrate specificity of an enzyme?
2 If an enzyme has the EC number 4.3.2.1,
What kind of enzyme does it belong to?
3 What is the optimum pH for an enzyme?
4 Why does not the activity of an enzyme
increase when its substrate concentration is too
high in reaction system?
目录
5 What is a holoenzyme? What is an
apoenzyme? What are cofactors?
6 What are Km and Vm? How to get Km
and Vm of an enzyme ?
7 How to analyze enzyme inhibition ?
8 What are the differences between
competitive and noncompetitive
inhibition?
目录