Download Enzymes - دانشکده پزشکی

‫عید سعید قربان بر‬
‫شما مبارک‬
‫یلدای زیبا هم همینطور!‬
In the Name of Allah
TUMS
ENZYMES:
…Make Life on Earth Possible
Abolfazl Golestani, PhD
An Important Question:
• Why should we as medical students,
study and learn about the ENZYMS?
• For answer go to slide No. 55
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
3
Chemical reaction
A
Catalyst
Product(s)
Reactant(s)
A +B
B
Catalyst
B+C
Catalysts
•Increase the rate of a reaction
•Are not consumed in the reaction
•Can act repeatedly
What are some of the known catalysts?
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
Heat
Acid
Base
Metals
4
Enzyme is either a pure protein or
may require a non-protein portion
Apoenzyme = protein portion
Apoenzyme + non-protein part = Holoenzyme
According to Holum, the non-protein portion may
be:
 A coenzyme - a non-protein organic substance
which is loosely attached to the protein part
 A prosthetic group - an organic substance which
is firmly attached to the protein or apoenzyme
portion
 A cofactor - these include K+, Fe++, Fe+++, Cu++,
Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
5
Basic enzyme reactions
S+EE+P
S = Substrate P = Product E = Enzyme
Swedish chemist Savante Arrhenius in 1888
proposed:
Substrate and enzyme form some
intermediate known as the EnzymeSubstrate Complex (ES):
S + E  ES Binding step
ES  P + E Catalytic step
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
6
There are two models of enzyme substrate
interaction
1. Lock and key model; Emil Fischer (1890)
2. Induced fit model; Daniel Koshland (1958)
5/23/2017
The active site:
• Substrate Binding Site
• Catalytic Site
Enzymes; by: Dr. Abolfazl
Golestani, PhD
7
Induced fit in Carboxypeptidase A
Three amino acids are located near the
active site (Arg 145, Tyr 248, and Glu 270)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
8
Enzyme-Substrate complex is
transient
S+E
S
E
P+E
When the enzyme unites with the
substrate, in most cases the forces that
hold the enzyme and substrate are noncovalent.
Binding forces of substrate are:
 Ionic interactions: (+)•••••(-)
 Hydrophobic interactions: (h)•••••(h)
 H-bonds: O-H ••••• O, N-H ••••• O, etc.
 van der Waals interactions
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
9
Some important characteristics of
enzymes
-Potent (high catalytic power) High reaction rates
They increase the rate of reaction by a factor of 103-1012
-Efficient (high efficiency)
catalytic efficiency is represented by Turnover number:
moles of substrate converted to product per second
per mole of the active site of the enzyme
-Milder reaction conditions Enzymatically catalyzed reactions occur
at mild temperature, pressure, and nearly neutral pH (i.e.
physiological conditions)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
10
Some important characteristics of
enzymes, cont.
-Specific (specificity)
Substrate specific
Reaction Specific
Stereospecific
-Capacity for regulation
Enzymes can be activated or inhibited so that the rate of
product formation responds to the needs of the cell
-Location within the cell
Many enzymes are located in specific organelles within the
cell. Such compartmentalization serves:
to isolate the reaction substrate from competing reactions,
to provide a favorable environment for the reaction, and
to organize the thousands of enzymes present in the cell
into purposeful pathways.
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
11
Specificity
Substrate Specificity
 Absolute specificity: For example Urease
 Functional Group specificity: For example
OH, CHO, NH2.
 Linkage specificity: For example Peptide
bond.
Reaction specificity
 Yields are nearly 100%
 Lack of production of by-products
 Save energy and prevents waste of
metabolites
Stereospecificity
 Enzymes can distinguish between
enantiomers and isomers
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
12
Enzymes requiring metal ions as
cofactors
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
13
Many Vitamins
vitamins are
are Coenzyme
coenzyme precursors
Many
precursors
• Many organism are unable to synthesize parts of the
coenzymes
• These parts must be present in the organism diet and
are called vitamins
Vitamin
Chemical Name
Biochemical Function
Coenzyme Chemistry
B1
Thiamine
Coenzyme
TPP
Decarboxylation of - keto
acids
B2
Riboflavin
Coenzymes FAD, FMN
Redox chemistry
Niacin
Nicotinamide
Coenzyme
NAD
Redox chemistry
B6
Pyridoxal
Coenzyme
PLP
Transamination reactions
B12
Cobalamine
Coenzyme B12
Radical rearrangements (lipid
degradation)
C
Ascorbic Acid
Coenzyme
Redox agent
( collagen formation)
H
5/23/2017
Biotin
Enzymes;Coenzyme
by: Dr. Abolfazl
Golestani, PhD
Carboxylation
14
Methods for naming enzymes
(nomenclature)
•
•
•
Very old method: Pepsin, Renin, Trypsin
Old method: Protease, Lipase, Urease
Systematic naming (EC = Enzyme
Commission number):
•
•
•
The name has two parts:
The first part: name of substrate (s)
The second part: ending in –ase, indicates the type of
reaction.
Additional information can follow in parentheses:
•
L-malate:NAD+ oxidoreductase (decarboxylating)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
15
Each enzyme has an EC number
= Enzyme Commission number
Enzyme
EC number
Alcohol dehydrogenase
Arginase
1.1.1.1
3.5.3.1
Pepsin
3.4.21.1
• EC number consists of 4 integers:
• The 1st designates to which of the six major classes an
enzyme belongs
• The 2nd integer indicates a sub class, e.g. type of bond
• The 3rd number is a subclassification of the bond type or
the group transferred in the reaction or both (a
subsubclass)
• The 4th number is simply a serial number
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
16
There are six functional classes of
enzymes
Class Names
Functions
1
Oxidoreductases AH + NAD+  A+ + NADH
2
Transferases
A-X + B  A + B-X
3
Hydrolases
A-OX + H2O  A-OH + HOX
4
Lyases
R1R2R3CCR4R5R6 
R1R2C=CR4R5 + R3 + R6
5
Isomerases
trans  cis, L-form D-form, etc.
6
Ligases
Formation of C-C, C-S, C-O, C-N
bonds by condensation reactions
coupled to ATP hydrolysis
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
17
EC3 Hydrolases
Function
EC 3.1
Acting on ester bonds
EC 3.2
Glycosylases
EC 3.3
Acting on ether bonds
EC 3.4
EC 3.5
EC5 Isomerases
Function
EC 5.1
Racemases and
epimerases
Acting on peptide bonds
(peptidases)
EC 5.2
cis-transIsomerases
Acting on carbon-nitrogen
bonds, other than peptide
bonds
EC 5.3
Intramolecular
isomerases
EC 5.4
Intramolecular
transferases
(mutases)
EC 5.5
Intramolecular
lyases
EC 5.99
Other isomerases
EC 3.6
Acting on acid anhydrides
EC 3.7
Acting on carbon-carbon
bonds
EC 3.8
Acting on halide bonds
EC 3.9
Acting on phosphorusnitrogen bonds
EC
3.10
Acting on sulfur-nitrogen
bonds
EC5/23/2017
Acting on carbon-phosphorus
Enzymes; by: Dr. Abolfazl
Golestani, PhD
3.11
bonds
18
Enzyme Nomenclature and
Classification
EC Classification
Class
Subclass
Sub-subclass
Serial number
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
19
Example of Enzyme Nomenclature
• Common name(s):
Invertase, sucrase
• Systematic name:
-D-fructofuranoside fructohydrolase
(E.C. 3.2.1.26)
• Recommended name:
5/23/2017
Enzymes; by: Dr. Abolfazl
-fructofuranosidase
Golestani, PhD
20
Kinetic
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
21
Energy barrier = Free Energy of Activation
X
T*
Y
T = Transition state
(Ea)
Thermodynamics:
Type (Exergonic or Endergonic)
Kinetics:
How fast the reaction will proceed
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
22
Enzyme Stabilizes Transition State
What’s the difference? Many enzymes function
Enzymes;
by: Dr. Abolfazl
by5/23/2017
lowering the activation energy
of reactions.
Golestani, PhD
23
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
‫عید سعید و عظیم غدیربرشما‬
‫مبارک‬
‫ميالد حضرت امام هادي (ع) را تهنيت مي‬
‫گويم‪،‬‬
‫میالد حضرت مسیح (ع) را هم همین‬
‫طور!‬
EA = Activation energy; a barrier to the reaction
Can be overcome
by adding
energy.......
......or by
catalysis
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
25
Enzymes Are Complementary to Transition State
X
If enzyme just binds substrate
then there will be no further
reaction
Enzyme not only recognizes substrate,
5/23/2017
Enzymes;
Dr. Abolfazlstate, see also Enz01
but
also induces the formation
ofby:
transition
Golestani, PhD
26
Active Site Is a Deep Buried Pocket
Why energy required to reach transition state
is lower in the active site?
It is a magic pocket
+
CoE (1)
(4)
(3)
5/23/2017
(2)
(1) Stabilizes transition
(2) Expels water
(3) Reactive groups
(4) Coenzyme helps
Enzymes; by: Dr. Abolfazl
Golestani, PhD
27
Juang RH (2004) BCbasics
Active Site Avoids the Influence of Water
+
-
Preventing the influence of water sustains the formation of stable ionic bonds
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
28
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115
Enzyme Reaction Mechanism
o Consider for example the mechanism
of Chymotrypsin:
o Enz06
o Enz07
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
29
Modes of rate enhancement
 Facilitation of Proximity
 Increase the Effective concentration
 Hold reactants near each other in proper
orientation
 Strain, Molecular Distortion, and
Shape Change
 Put a strain on susceptible bonds
 General Acid –Base Catalysis
 Transfer of a proton in the transition state
 Covalent Catalysis
 Form covalent bond with substrate
destabilization of the substrate
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
30
Factors Affecting Rate of Enzyme Reactions






Temperature
pH
Enzyme concentration [E]
Substrate concentration [S]
Inhibition
Regulation (Effectors)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
31
1- Optimum Temperature
 Little activity at low temperature (low number of collisions)
 Rate increases with temperature (more successful collisions);
rate doubles for every 10°C increase in temperature
 Most active at optimum temperatures (usually 37 oC in humans)
 Enzymes isolated from thermophilic organisms display maxima
around 100 °C
 Enzymes isolated from psychrophilic organisms display maxima
around 10 °C.
 Activity lost with denaturation at high temperatures
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
32
2- Optimum pH
• Effect of pH on ionization of active site
• Effect of pH on enzyme denaturation
• Each enzyme has an optimal pH (~ 6 - 8 )
– Exceptions :
digestive enzymes in the stomach (pH 2)
digestive enzymes in the intestine (pH 8)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
33
3- Enzyme concentration
• The Rate (v) of reaction Increases proportional to
the enzyme concentration [E] ([S] is high)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
34
4- Substrate concentration
• When enzyme concentration is constant,
increasing [S] increases the rate of reaction,
BUT
• Maximum activity reaches when all E combines
with S (when all the enzyme is in the ES form)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
35
Enzyme
Velocity
Curve, see also
Enz02
0
1
2
3
4
5
6
7
8
S
+
E
80
60
Product (v)
40
(in a fixed
period of
time)
20
0
5/23/2017
P
0
2
4
6
Substrate (mole) [S]
Enzymes; by: Dr. Abolfazl
Golestani, PhD
8
36
Juang RH (2004) BCbasics
‫بسم هللا الرحمن الرحیم‬
Enzymes
rd
3 part
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
37
Michaelis-Menten Equation
S
K+1
E
k-1
S
E
k2
P
maximal velocity, Vmax
5
v, µmol/min
4
3
0.5Vmax
2
Km
1
0
0
5/23/2017
10
20
30
[S], mM
40
50
Enzymes; by: Dr. Abolfazl
Golestani, PhD
38
MM Equation Derivation (steady state)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
39
Practical Summary - Vmax and Km
• Vmax
– How fast the reaction can occur under ideal
circumstances
• Km
– Range of [S] at which a reaction will occur
– Binding affinity of enzyme for substrate
• LARGER Km  the WEAKER the binding affinity
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
40
Practical Summary,
cont.
Enzyme
Substrate
Km (mM)
Catalase
H2O2
1,100
Chymotrypsin
Gly-Tyr-Gly
108
Carbonic anhydrase
CO2
12
Beta-galactosidase
D-lactose
4
Acetylcholinesterase
acetylcholine (ACh)
0.09
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
41
Practical Summary;
cont.
• Kcat/Km
– Practical idea of the catalytic efficiency, i.e.
how often a molecule of substrate that is
bound reacts to give product
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
42
Order of Reaction
1. When [S] << Km
vo = (Vmax/Km )[S]
2. When [S] = Km
vo = Vmax/2
3. When [S] >> Km
vo = Vmax
5/23/2017
zero order
Mixed order
2
First order
Enzymes; by: Dr. Abolfazl
Golestani, PhD
43
Importance of Vi
in Measurement of Enzyme Activity
S
E
k1
k-1
k2
S
E
P
Working with vo minimizes complications with
1. reverse reactions
2. product Inhibition
The rate of the reaction catalyzed by an enzyme
in a sample is expressed in Units.
Units = V = activity = Micromoles (mol; 10-6 mol or ….),
of substrate reacting or product produced per min.
It is better to measure it at linear part of the curve
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
44
Lineweaver-Burk plot
1
vo
-1
Km
vo
1/2
1
Vmax
1
Km 1
1


v Vmax [S] Vmax
5/23/2017
Km Direct plot S
1/S
Vmax [S]
v
Km  [S]
Enzymes; by: Dr. Abolfazl
Golestani, PhD
45
Juang RH (2004) BCbasics
Double reciprocal plot
Allosteric Enzymes
• Why the sigmoid shape?
• Allosteric enzymes are multi-subunit enzymes,
each with an active site
• They show a cooperative response to substrates
• See Enz13
hyperbolic curve;
Michaelis-Menten
kinetics
Sigmoidal curve
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
46
 Irreversible Inhibition=Enzyme
Stops Working Permanently
1.
2.
Destruction of enzyme
Irreversible Inhibitor=forms covalent bonds to E
(inactive E)
Examples:
–
Diisopropylfluorophosphate
• inhibits acetylcholine esterase
• binds irreversibly to –OH of serine residue
–
Cyanide and sulfide
• Inhibit cytochrome oxidase
• bind to the iron atom
–
Fluorouracil
• inhibits thymidine synthase (suicide inhibition - metabolic
product is toxic )
–
Aspirin
• Inhibits prostaglandin synthase
• acylates an amino group of the cyclooxygenase
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
47
 Reversible Inhibition=Temporary
Decrease of Enzyme Function
•
Three types based on “how increasing [S]
affects degree of inhibition”:
1. Competitive: degree of inhibition
decreases
2. Non-competitive: degree of inhibition is
unaffected
3. Anti- or Uncompetitive: degree of
inhibition increases
 The Lineweaver-Burk plot is useful in
determining the mechanisms of actions of
various inhibitors, see Enz04
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
48
The Effects of Enzyme Inhibitors
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
49
Example
• When a slice of apple is exposed to air, it quickly
turns brown. This is because the enzyme
o-diphenyl oxidase catalyzes the oxidation of
phenols in the apple to dark-colored products.
• Catechol can be used as the substrate. The
enzyme converts it into o-quinone (A), which is
then further oxidized to dark products.
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
50
Experiments
No Inhibitor
Tube A
Tube B
Tube C
Tube D
[S]
4.8 mM
1.2 mM
0.6 mM
0.3 mM
1/[S]
0.21
0.83
1.67
3.33
Δ OD540
(Vi)
0.081
0.048
0.035
0.020
1/Vi
12.3
Tube A
[S]
4.8 mM
20.8
Tube B
1.2 mM
31.7
Tube C
0.6 mM
Tube A
Tube B
Tube C
Tube D
[S]
4.8
mM
1.2
mM
0.6
mM
0.3
mM
1/[S]
0.21
0.83
1.67
3.33
ΔOD540
(Vi)
0.060
0.032
0.019
0.011
1/Vi
16.7
31.3
52.6
90.9
50.0
Tube D
0.3 mM
1/[S]
0.21
0.83
1.67
3.33
ΔOD540
(Vi)
0.040
0.024
0.016
0.010
1/Vi
25
41
62
102
5/23/2017
effect of para-hydroxybenzoic
acid (PHBA)
effect of phenylthiourea
Enzymes; by: Dr. Abolfazl
Golestani, PhD
51
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
52
I- Competitive Inhibition
EI
S
Competitive
V [S]
v  max
Km  [S]
CI
V [S]
v  max
K m  [S]
Ki
E
Sc + E
+
I
ES
1 Km 1
1


v Vmax [S] Vmax
5
E+P
1 K m 1
1


v Vmax [S] Vmax
2.5
No I
4
v, µmol/min
µmol/min
v,
0.5V
max
+CI
0.5V
max
3
2
K
m
Kmapp
1
-1/Km
app
+CI
2
1/v, /µmol/min
/µmol/min
1/v,
 [I] 

  1
 Kic 
-1/Km
-1/K
m
Km
Kmapp/Vmax
1.5
1
0.5
0
K /V
K m
/V max
m max
No I
1/V 1/V
max max
0
0
5/23/2017
10
20
30
[S], mM
40
50
-0.6 -0.4 -0.2
Enzymes; by: Dr. Abolfazl
Golestani, PhD
0 0.2 0.4 0.6 0.8
1/[S], /mM
1
53
II- Noncompetitive Inhibition
S
Noncompetitive
(mixed-type)
NCI
S
V [S]
v  max
Km  [S]
E
Vmax [S]
v
K m   ' [S]
EI
E
Kic
S+E
+
I
NCI
1 Km 1
1


v Vmax [S] Vmax
55
E+P
1 K m 1
'


v Vmax [S] Vmax
2.5
44
0.5Vmax
33
+ NCImax
0.5V
0.5Vmax
22
K
11
Km
 [I] 

  1
 Kic 
2
1/v, /µmol/min
/µmol/min
1/v,
No I
v, µmol/min
ESI
Kiu
ES
+
I
 [I] 

'  1
 Kiu 
-1/K m
-1/K
m
m
1.5
1/Vmaxapp
Km/Vmaxapp
+ NC I
1
0.5
K K/V /V
m mmax
max
1/V
max
1/V
00
No I
max
0
00
5/23/2017
10
10
20
20
30
30
[S],
[S], mM
mM
40
40
50
50
-0.6 -0.4 -0.2
Enzymes; by: Dr. Abolfazl
Golestani, PhD
0 0.2 0.4 0.6 0.8
1/[S], /mM
1
54
III- Uncompetitive Inhibition
Uncompetitive
(catalytic)
Vmax [S]
v
Km  [S]
S
E
Vmax [S]
v
Km   ' [S]
ESI
Kiu
UCI
S+E
ES
E+P
+
I
1 Km 1
1
1 Km 1
'




v Vmax [S] Vmax
v Vmax [S] Vmax
5
2.5
No I
4
2
max
+ UC I
0.5V
max
3
Km
2
K
1
0.5Vmax
 [I] 

'  1
 Kiu 
app
m
-1/Km
Km
-1/Km
app-1/Km
1/v, /µmol/min
v, µmol/min
0.5V
1/Vmaxapp
1.5
Kmapp/Vmaxapp
1
0.5
1/Vmax
K /V
m max
+ UC I
Km/Vmax
No I
1/Vmax
0
0
0
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10
20
30
[S], mM
[S].
40
50
-0.6 -0.4 -0.2
Enzymes; by: Dr. Abolfazl
Golestani, PhD
0 0.2 0.4 0.6 0.8
1/[S]. /mM
1/[S],
1
55
Enzyme Inhibitors in Medicine
• Many current pharmaceuticals are enzyme inhibitors
(e.g. HIV protease inhibitors for treatment of AIDS)
• An example: Ethanol is used as a competitive
inhibitor to treat methanol poisoning
 Methanol
Alcohol dehydrogenase
formaldehyde (very toxic)
 Ethanol competes for the same enzyme
 Administration of ethanol occupies the enzyme
thereby delaying methanol metabolism long enough
for clearance through the kidneys
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
56
Some diagnostically important enzymes
Aminotransferases
Aspartate aminotransferase
(AST or SGOT)
Alanine aminotransferase
(ALT, or SGPT)
Myocardial infarction
Viral hepatitis
Lactate Dehydrogenase (LDH)
myocardial infarction
Creatine Kinase (CK)
Myocardial infarc., brain,
skeletal muscle disorder
Cholinesterase
Liver, erythrocytes
Gamma-glutamyltransferase
Liver damage
Acid phosphatase
Carcinoma of prostate
Alkaline phosphatase (AP)
Bone disease
Lipase
Acute pancreatitis
Ceruloplasmin
Hepatolenticular degeneration
(wilson’s disease)
Alpha-amylase
Intestinal obstruction
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
58
5/23/2017
Useful enzymes for
early diagnosis of
dental caries and
periodontal disease
Enzymes; by: Dr. Abolfazl
Golestani, PhD
59
Isozymes of Lactate Dehydrogenase
Isozymes:
– Are catalitically identical (have same catalytic activity) BUT
physically distinct
– Can be detected by gel electrophoresis (different electrical charge)
– Occur in oligomeric enzymes like lactate dehydrogenase (LDH)
In LDH
• Protomers H and M can combine to make five different
tetramers.
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
60
Isoenzymes of Creatine Kinase
• CK has 3 forms dimer B
and M chains:
• CK1= BB
• CK2= MB
• CK3=MM
• Heart, the only tissue
rich in CK2, increases
4-8 hr after chest
pains- peaks at 24 hr.
• LDH peaks 2-3 days
after MI.
• New markers:
Troponin T, Troponin I
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
61
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
62
5- Regulation (Effectors)
Effectors can be classified as follows:
According to type:
• Homotropic effector: Substrate itself is the effector
• Heterotropic effector: substance other than substrate
is the effector
According to their effect:
• Activators (positive effectors)
– Increase the rate of enzyme
• Inhibitors (negative effectors)
– Decrease the velocity of reaction
– Stop the enzyme
Increase or decrease
in enzyme reaction
rate is reflected in the
graph of V versus S
• Irreversible
• Reversible
– Competitive
– Non-competitive
5/23/2017– Uncompetitive
Enzymes; by: Dr. Abolfazl
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63
Metabolic Pathways
• A metabolic pathway is a chain of
enzymatic reactions
– Most pathways have many steps, each having
a different enzyme (E1, E2, E3, E4)
– Step by step, the initial substance used as
substrate by the first enzyme is transformed
into a product that will be the substrate for the
next reaction
• Metabolic regulation is necessary to:
– maintain cell components at appropriate levels.
– conserve materials and energy.
5/23/2017
Enzymes; by: Dr. Abolfazl
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64
Regulation of “Enzyme Activity”
A. Regulation at transcription level
(slowest)
B. Isozymes: enzymes specific for distinct
tissues and developmental stages
C. Compartmentation of S, E and P
D. Specific proteolytic cleavage
E. Covalent modification
(Reversible phosphorylation or adenylation)
F. In response to metabolic products
(fastest)
1.
2.
3.
4.
Substrate level control
Product Inhibition
Feedback control
Allosteric Effectors
5/23/2017
Enzymes; by: Dr. Abolfazl
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65
A. Regulation at Transcription Level
1. Regulation of [E] by
• Gene repression
• Induction of genetic expression of
enzyme
2. There is competition in a cell
between the processes of protein
synthesis and protein destruction
• By altering these rates, one can alter
the whole cell catalytic rate
3. It is rather slow
5/23/2017
Enzymes; by: Dr. Abolfazl
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66
B. Isoenzymes
• Isozymes provide a means of
regulation, specific to distinct
tissues and developmental
stages
• Differential expression of
isozymes
• LDH (for example)
• Preferential substrate affinity
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Enzymes; by: Dr. Abolfazl
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67
C. Compartmentalization of enzymes
Substrates and cofactors within the cell
are also compartmentalized
Examples:
• Enzymes of glycolysis are located in
the cytoplasm
• Enzymes of citric acid cycle are in the
mitochondria
• Hydrolytic enzymes are found in the
lysosome
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68
D. Proteolytic activation
Activation of a zymogen
• Some enzymes are secreted as inactive
precursors, called zymogens.
• Pancreatic proteases - trypsin,
chymotrypsin, elastase, carboxypeptidase
are all synthesized as zymogens:
trypsinogen, chymotrypsinogen,
proelastase and procarboypeptidase
• Clotting factors are also part of a proteolytic
cascade
• Hormone peptides (Pro-insulin
Insulin)
• An on/off switch more than regulation
5/23/2017
Enzymes; by: Dr. Abolfazl
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69
E. Covalent modification
Reversible phosphorylation
Phosphorylation is the most common type
of modification. Two important classes of
enzymes are:
– Kinases
Add a phosphate group to
another protein/enzyme (phosphorylation)
• transfer of phosphoryl group from ATP
to -OH group of serine, threonine or
tyrosine
– Phosphatases
Remove a phosphate
group from a protein/enzyme
(dephosphorylation)
5/23/2017
Enzymes; by: Dr. Abolfazl
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70
1- Control of [S]
• Concentration of substrate and
product also control the rate of
reaction, providing a biofeedback
mechanism
• Usually: 0.1 Km<[SPhysiologic]<10 km
Mild changes
in [S]
Change in enzyme
activity
Homotropic effectors – substrate itself
(binding at different site other than the active
site) affects enzyme activity on other substrate
molecules. Most often this is a positive effector.
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Enzymes; by: Dr. Abolfazl
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71
2- Product inhibition
• Enzyme is reversibly inhibited by the product
Example: hexokinase in the first reaction of glycolysis is
inhibited by glucose-6-phosphate (G6P; the product)
glucose + ATP
glucose-6-phosphate + ADP
_
Why?
As v approaches Vmax, the product becomes significant, and can
compete with the substrate for the enzyme.
The product becomes a competitive inhibitor and slows down activity
of the enzyme.
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
72
3- Negative feedback control
(end product inhibition)
• Final product of a metabolic sequence feeds-back
negatively on early steps
• In feedback inhibition, there is a second binding site on the
enzyme where the inhibitor binds, so that the inhibitor is
not necessarily similar in structure to the substrate
Enz 1
A
_
Enz 2
B
Enz 3
C
Enz 4
D
E
What happens?
•
•
•
As the need for product E decreases, E will accumulate
Most efficient to inhibit at first step of the pathway, slow the first
reaction so intermediates do not build up
An increase in the concentration of E, leads to a decrease in its rate
of production of E
5/23/2017
Enzymes; by: Dr. Abolfazl
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73
Regulation of the metabolism, feed-back
inhibition by the final product - end product
inhibition
1. Simple feed-back inhibition. The
final product (E) inhibits the step
from A to B.
2. Co-operative feed-back
inhibition. Both final products (D,
E) inhibit the first step of their own
synthesis together.
3. Multivalent feed-back inhibition.
5/23/2017
4. Inhibition at a ramification of a
biosynthesis pathway (sequential
inhibition)
74
Enzymes; by: Dr. Abolfazl
Golestani, PhD
4- Positive feedforward control
• Earlier reactants in a metabolic sequence
feed-forward positively on later steps.
+
If A is accumulating, it
speeds up downstream
reactions to use it up
+
Metabolism involves
the complex integration
of many feedback and
feedforward loops
5/23/2017
Enzymes; by: Dr. Abolfazl
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75
4- Allosteric control
• Allosteric activator stabilizes active "R" state
– shift the graph to the left
• Allosteric inhibitor stabilizes less active or inactive "T" state
– shift the graph to the right
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
76
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Enzymes; by: Dr. Abolfazl
Golestani, PhD
77
Multi reactant enzymes reactancy
• Published by W. W. Cleland in1963
• Nomenclature is based on number of
substrates and products in the
reaction.
• Reactancy: the number of kinetically
significant substrates or products and
designated by syllables Uni, Bi, Ter,
Quad.
5/23/2017
AP
Uni Uni
AP+Q
Uni Bi
A+BP+Q
Bi Bi
A+B+CP+Q+R+S
Ter Quad
Enzymes; by: Dr. Abolfazl
Golestani, PhD
78
Multi reactant enzymes mechanism
 Sequential - if all S add to E before any P are
released.
– Sequential ordered - if S add in an
obligatory order (two on; two off)
– Sequential random - if S do not add in
obligatory order (two on; two off)
 Ping Pong - If one or more S released before
all S bind
• (one on, one off; one on, one off);
• Note: there is some sort of modified
enzyme intermediate (often covalent
intermediate)
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
79
5/23/2017
Enzymes; by: Dr. Abolfazl
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80
Random sequential (example)
5/23/2017
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Ordered sequential (example)
5/23/2017
Enzymes; by: Dr. Abolfazl
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82
Ping pong or double displacement
mechanism
5/23/2017
Enzymes; by: Dr. Abolfazl
Golestani, PhD
83
Double displacement (example)
5/23/2017
Enzymes; by: Dr. Abolfazl
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84
Other kinds of enzymes
• Some ribonucleoprotein enzymes
have been discovered
–The catalytic activity is in the
RNA part
–They are called Ribozymes
• Catalytic antibodies are called
Abzymes
5/23/2017
Enzymes; by: Dr. Abolfazl
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85
‫موفق باشید‬
5/23/2017
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