ENZYMES
Prof Francis J. Mulaa
Dept Biochemistry 2017
An Important Question:
• Why should we as medical students,
study and learn about the ENZYMS?
• For answer go to slide No. 55
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
2
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?
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
Heat
Acid
Base
Metals
3
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+++
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
4
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
5
There are two models of enzyme substrate
interaction
1. Lock and key model; Emil Fischer (1890)
2. Induced fit model; Daniel Koshland
(1958)
01/20/21
The active site:
• Substrate Binding Site
• Catalytic Site
Enzymes; by: Prof. Francis J. Mulaa,
PhD
6
Induced fit in Carboxypeptidase A
Three amino acids are located near the
active site (Arg 145, Tyr 248, and Glu 270)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
7
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
8
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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
9
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.
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
10
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
11
Enzymes requiring metal ions as
cofactors
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
12
Many vitamins are coenzyme precursors
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
13
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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
14
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
15
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
01/20/21
Enzymes; by: Prof. Francis J.
Mulaa, PhD
16
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
EC01/20/21Acting on carbon-phosphorus
Enzymes; by: Prof. Francis J.
Mulaa, PhD
3.11
bonds
17
Enzyme Nomenclature and
Classification
EC Classification
Class
Subclass
Sub-subclass
Serial number
01/20/21
Enzymes; by: Prof. Francis J.
Mulaa, PhD
18
Example of Enzyme Nomenclature
• Common name(s):
Invertase, sucrase
• Systematic name:
-D-fructofuranoside fructohydrolase
(E.C. 3.2.1.26)
01/20/21
• Recommended name:
Enzymes; by: Prof. Francis J. Mulaa,
-fructofuranosidase
PhD
19
Kinetic
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
20
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
21
Enzyme Stabilizes Transition State
What’s the difference? Many enzymes function
Enzymes; by: Prof. Francis J. Mulaa,
by01/20/21
lowering the activation energy
of reactions.
PhD
22
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
ENZYMES
Prof Francis J. Mulaa
Dept Biochemistry 2008
EA = Activation energy; a barrier to the reaction
Can be overcome
by adding
energy.......
......or by
catalysis
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
24
Enzymes Are Complementary to Transition State
X
If enzyme just binds substrate
then there will be no further
reaction
Enzyme not only recognizes substrate,
Enzymes; by: Prof. Francis J. Mulaa,
01/20/21
but
also induces the formation
of transition
state, see also Enz01
PhD
25
Active Site Is a Deep Buried Pocket
hy energy required to reach transition sta
lower in the active site?
It is a magic pocket
+
CoE (1)
(4)
-
(3)
01/20/21
(1) Stabilizes transitio
(2) Expels water
(2)
(3) Reactive groups
(4) Coenzyme helps
Enzymes; by: Prof. Francis J. Mulaa,
PhD
26
Juang RH (2004) BCbasics
Active Site Avoids the Influence of Water
+
Preventing the influence of water sustains the
Prof. Francis J. Mulaa,
01/20/21
formation
of stable Enzymes;
ionic by:
bonds
PhD
27
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
28
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
Enzymes; by: Prof. Francis J. Mulaa,
01/20/21
PhD
29
Factors Affecting Rate of Enzyme Reactions






Temperature
pH
Enzyme concentration [E]
Substrate concentration [S]
Inhibition
Regulation (Effectors)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
30
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
31
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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
32
3- Enzyme concentration
• The Rate (v) of reaction Increases proportional to
the enzyme concentration [E] ([S] is high)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
33
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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
34
Enzyme
Velocity
Curve, see also
Enz02
0
1
2
3
4
5
6
7
8
80
Product (v)
60
01/20/21
P
40
Con
20
0
S
+
E
0
2
E]
[
t
n
sta
4
6
Substrate (mole) [S]
Enzymes; by: Prof. Francis J. Mulaa,
PhD
(in a
fixed
period
of time)
8
35
Juang RH (2004) BCbasics
Enzymes
Enzymes
3rd part
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
36
Michaelis-Menten Equation
S
K+1
E
k-1
S
E
k2
P
maximal velocity, Vmax
0.5Vmax
Km
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
37
MM Equation Derivation (steady state)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
38
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
39
Practical Summary, cont.
Enzyme
Substrate
Km (mM)
Catalase
H2 O 2
1,100
Chymotrypsin
Gly-Tyr-Gly
108
Carbonic anhydrase
CO2
12
Beta-galactosidase
D-lactose
4
Acetylcholinesterase
acetylcholine (ACh)
0.09
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
40
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
01/20/21
Enzymes; by: Prof. Francis J.
Mulaa, PhD
41
Order of Reaction
1. When [S] << Km
vo = (Vmax/Km )[S]
zero order
2. When [S] = Km
vo = Vmax/2
3. When [S] >> Km
vo = Vmax
01/20/21
Mixed order
2
First order
Enzymes; by: Prof. Francis J. Mulaa,
PhD
42
Importance of Vi
in Measurement of Enzyme Activity
S
E
k1
k-1
S
E
k2
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
43
Lineweaver-Burk plot
1
vo
1/2
1
Vmax
Double reciprocal plot
1/S
1
Km 1
1
=
+
v Vmax [S] Vmax
01/20/21
Km Direct plot S
Vmax [S]
v=
Km + [S]
Enzymes; by: Prof. Francis J. Mulaa,
PhD
44
Juang RH (2004) BCbasics
-1
Km
vo
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
45
 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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
46
 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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
47
The Effects of Enzyme Inhibitors
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
48
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.
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
49
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
01/20/21
effect of para-hydroxybenzoic
acid (PHBA)
effect of phenylthiourea
Enzymes; by: Prof. Francis J. Mulaa,
PhD
50
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
51
I- Competitive Inhibition
EI
S
Competitive
V [S]
v = max
Km + [S]
,
in v
l/m
o
,v
1
2
3
[S], mM
20
30
10
40
S+E
+
I
1 Km 1
1
=
+
v Vmax [S] Vmax
50
m
m 0.5
.5
-1
/K
-1
/K
m
K
x
0+
.5
VIma
app
C
max
0.5V
Km
-1/Kmapp
01/20/21
1 aK m 1
1
=
+
v Vmax [S] Vmax
1
Kmapp/Vmax
a
xmax
1
/V
1/Vm
m max
K m
/V max
K /V
No I
1.5
.5
2
4
5
E+P
1/[S], /mM
-0.6 -0.4 -0.2 0 0..2
2 0.4
.4 0.6 0.8
.8 1
0
æ [I] ö
÷
a = ç1+
è Kic ø
Km
ES
,1
/v
1
,
/v
0
V [S]
v = max
aK m +[S]
E
l/in
o
m
l/in
o
m
0
CI
Kic
+C I
No I
2.5
.5
Enzymes; by: Prof. Francis J. Mulaa,
PhD
52
II- Noncompetitive Inhibition
in v
l/m
o
,
1
1
10
0
m
K
v=
Vmax [S]
aK m + a' [S]
[[S
S]],,m
mM
M
2
20
3
30
Km
4
40
0
0.5Vmax
+
0.5
NCI
Vmax
01/20/21
ESI
Kiu
ES
+
I
E+P
1 Km 1
1
=
+
v Vmax [S] Vmax
1 aK m 1
a'
=
+
v Vmax [S] Vmax
1/[S], /mM
-0.6 -0.4 -0.2 0 0..2
2 0.4 0.6 0.8 1
0
app
1/Vmax
1/Vmax
æ [I] ö
÷
a = ç1+
è Kic ø
m
m
-1
-1/K
/K
0..5
5
1
1..5
5
0.5Vmax
4
5
50
0
NCI
æ [I] ö
÷
a' = ç1+
è Kiu ø
2
3
E
Kic
S+E
+
I
,1
/v
,
/v
1
0
0
0
Vmax [S]
Km + [S]
S
EI
E
in
l/m
o
l/in
o
m
Noncompetitive
(mixed-type)
v=
NCI
S
NoI
Kax/V
m
1/V m max
NoI
m
m
m
m
a
xax
K
/
V
K /V
app
+NC I
2
2..5
5
Enzymes; by: Prof. Francis J. Mulaa,
PhD
53
III- Uncompetitive Inhibition
0
10
Vmax [S]
v=
Km + a' [S]
[S]., mM
20
30
40
0
l/in v
o
m
,
1
m
K
m
K
2
Kmapp
3
max
0
.5
V
+
U
CI
0.5Vmax
4
5
01/20/21
E
50
1/[S].
/mM
1
/[S], /m
M
-0.6
-0.4
-0.2
0 0
0.2
0.4
0.6
0.8
1
-0
.6 -0
.4 -0
.2 0
.2 0
.4 0
.6 0
.8 1
0
0
max
app
1/V
-1/Km
m
-1/K
0.5Vmax
æ [I] ö
÷
a' = ç1+
è Kiu ø
-1/Kmapp
No I
1/Vmax
0.5
0
.5
,1
/v
,
/v
1
Vmax [S]
v=
Km + [S]
S
in
l/m
o

m
Uncompetitive
(catalytic)
ESI
Kiu
UCI
S+E
ES
E+P
+
I
1 Km 1
1
1 Km 1
a'
=
+
=
+
v Vmax [S] Vmax
v Vmax [S] Vmax
1
1
max
1/V
app
Kmapp/V
mmax
max
No I
K /V
+UC I
m max
K /V
1.5
1
.5
2
2
2.5
2
.5
Enzymes; by: Prof. Francis J. Mulaa,
PhD
54
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
toxic)
Alcohol dehydrogenase
formaldehyde
(very
 Ethanol competes for the same enzyme
 Administration of ethanol occupies the enzyme
thereby delaying methanol metabolism long enough
for clearance through the kidneys
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
55
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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PhD
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01/20/21
Useful enzymes for
early diagnosis of
dental caries and
periodontal disease
Enzymes; by: Prof. Francis J. Mulaa,
PhD
58
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.
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
59
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 48 hr after chest painspeaks at 24 hr.
• LDH peaks 2-3 days
after MI.
• New markers:
Troponin T, Troponin I
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PhD
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01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
61
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
• Irreversible
• Reversible
Increase or
decrease in enzyme
reaction rate is
reflected in the
graph of V versus
S
– Competitive
01/20/21 – Non-competitive Enzymes; by: Prof. Francis J. Mulaa,
PhD
– Uncompetitive
62
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.
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PhD
63
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.
01/20/21
Substrate level control
Product Inhibition
Feedback control
Allosteric Effectors
Enzymes; by: Prof. Francis J. Mulaa,
PhD
64
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
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PhD
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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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PhD
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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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PhD
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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
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PhD
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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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
69
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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PhD
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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.
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
71
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
_
B
Enz 2
C
Enz 3
D
Enz 4
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
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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.
01/20/21
4. Inhibition at a ramification of a
biosynthesis pathway (sequential
Enzymes; by: Prof. Francis
J. Mulaa,
73
inhibition)
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
74
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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PhD
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01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
76
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.
AP
Uni Uni
AP+Q
Uni Bi
A+BP+Q
Bi Bi
A + B + C  P + Q + R + S Ter Quad
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
77
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)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
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01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
79
Random sequential (example)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
80
Ordered sequential (example)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
81
Ping pong or double displacement
mechanism
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
82
Double displacement (example)
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
83
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
01/20/21
Enzymes; by: Prof. Francis J. Mulaa,
PhD
84