MODULATION OF ENZYME ACTIVITY
Effectors
Inhibitor: Activator:
decreases increases
reaction rate reaction rate
vi va
Degree of inhibition: Degree of activation:
0 0
i i
v v
ε = v− 0
0 a a
v v ε = v−
INHIBITION
REVERSIBLE IRREVERSIBILE
distinction:
Vmax
KONTROLL
+REVERZIBILIS INHIBITOR
RREVERZIBILIS INHIBITOR
E S ES E P
EI
+ ⇋ → +
⇃↾
S 2
K ES k
E S E P
EI
+ ←→ → +
↓
Competitive inhibition
Competition between S and I for the active sites of the enzyme, or mutual exclusion
I may be an:
substrate analogue alternative substrate product
MODEL 1.: Classical competitive inhibition:
I competes with S for occupation of the same active site
S I
3
COMPETITIVE INHIBITION
MODEL 2.: steric hindranceA
I S
Binding of I to another site sterically hinders S in binding to the active site of enzyme.
COMPETITIVE INHIBITION
5
MODEL 3.: steric hindrance B
An analog part of S and I compete for a common binding site.
S I
E
MODEL 4.: overlaping
Sites 1 and 3 can bind I, 2 and 4 can bind S, but both
I S
1 2 3 4
COMPETITIVE INHIBITION
COMPETITIVE INHIBITION
MODEL 5.:
Binding of I changes the conformation of the enzyme which prevents binding of S to the active centre.
End product inhibition (feed back inhibition) is typical example of this case.
S I S
S I
I
7
Kinetics of competitive inhibition
Basic equations for competitive inhibition:
if kapp>0 than I is an alternative substrate
if kapp=0 than I is a „dead end” competitive inhibitor
S 2
K ES k
app
i k
E S E P
I K
EI E P
+ ←→ → + +
→ + ′ վ
( )
s
K E S ES
= ⋅
( )
i
K E I EI
= ⋅
Kinetics of competitive inhibition
Alternative substrate: the enzyme is able to transform the structural analogous molecule, too. → an alternative product is formed.
Enzymes with group and region specifity have numerous al- ternative substrates
Example: the enzymes of liver: alcohol dehydrogenase, alde- hyde dehydrogenase:
E + S ' ← → E + P'
9
Kinetics of competitive inhibition
Repeat the deduction:
product formation rate:
Mass balance of enzyme:
S 2
K k
ES
app
i k
E S E P
I K
EI E P
+ ←→ → + +
→ + ′ վ
( )
s
K E S ES
= ⋅
( )
i
K E I EI
= ⋅
V dP
dt k (ES)2
= =
E
0= + E (E S) + (E I)
Kinetics of competitive inhibition
Divide the two equation:
Substitute:
2 o
V k (ES) E =E (ES) + (EI)
+ ( )
s
K E S ES
= ⋅
( )
i
K E I EI
= ⋅
2 s o
s I
k E S
V K
S I
E E E E
K K
= + + 2 1
s o
s I
S
V K
S I k E
K K
= + +
V
max= k E
2 o11
Kinetics of competitive inhibition
Simplified forms of reaction rate:
or:
or:
max 1
s i
V S
V I
K S
K
=
+ +
max s 1
i
V V S
K I S
K
=
+ +
max i
I S
I
v ( S )
v K ( I )
K ( S )
K
= + +
Kinetics of competitive inhibition
13
S 1 K 1 I V
K V
1 V 1
I max
S max
+
+
=
HC C NH2H H
H
C HO O
L-alanin
O C H H
NH2 HC C
O NH
cikloszerin
Competitive inhibition
Alternative substrates: for hexokinase: glucose, fructose
S-analogons: drugs:
Effect of sulfami- des (antimicrobial drugs): substrate analogon act as competitive inhi- bitor.
Competitive inhibition
15
K S 1 I K V S V
i s max
+
+
=
K S 1 P K V S V
P s
max
+
+
=
1
1 1max
2
S1 1
S2
V V S
K 1 S S
K
=
+ +
2
2 2max
1
S2 2
S1
V V S
K 1 S S
K
=
+ +
Analogous inhibitions
competitive inhibition: product inhibition:
alternative or competing substrates
Noncompetitive inhibition
17
Noncompetitive inhibition
Inhibitor binds to an other active site of the enzyme and does not affect the binding of the substrate – does not change the affinity of the enzyme to the substrate.
It exists only when rapid equilibrium can be supposed, Ks=Km.
Equations of noncompetitive inhibition:
Ks
kp
E + S ES E + P
+ +
I I
K K K
K E.S
ES EI.S
ESI és K E.I EI
ES.I
s= = i= = ESI
V ES
=
V = kp(ES)
V V
ES
E ES EI ESI
max
= + + +
V V
S K
1 S
K I K
S.I K K vagy
V V
S
K 1 I
K S 1 I
K illetve
V = V 1
1 I K
S
K S
max
s
s i s i
max s
i i
max
i s
= + + +
=
+
+ +
+
+
V V S
K S ahol V V 1 1 I
K
maxi s
maxi max
i
= + =
+
where or
or
Noncompetitive inhibition
Inhibitor changes the value of the apparent Vmax, but does not change the values of Ks( or Km).
19
V 1/V
}
11 Vmaxi
1 I Ki Vmax
=
+ K
V 1 I K
m
max i
+
K V
m max
I Vmax
Vmaxi
I
Noncompetitive inhibition
The inhibitor affects the apparent Vmaxvalue but does not change Ks(or Km).
Noncompetitive inhibition
Examples:
H+ions’ effect on chymotripsine. Here a proton acceptor site exists in the active centre, which can be inhibited by increasing H+-ion concentration. (L-B plot shows clear noncompetitive inhibition, (but do not forget the complex effect of the pH on enzymes).
Heavy metal molecules(-SH reagensek), or cyanides.
Often these effects are irreversible.
21
Noncompetitive inhibition
Surface of slices apple gets brown in air: o-diphenol oxidase enzyme catalyses the catechol→ o-quinone reaction
this and other reac- tion products give the brown color
competitive inhibitor of o-diphenol oxidase is para-hydroxy- benzoic acid (PHBA), a structural analog.
Noncompetitive inhibition
competitive inhibitor of o-diphenol oxidase is para-hydroxybenzoic acid (PHBA), a structural analogon
noncompetitive inhibitor is: phenyl- thiourea, bound to copper ion what is necessary to enzyme activity.
23
Uncompetitive inhibition
Fixed order: the inhibitor must join second, after the substrate
1 V
K V
1 S
1
V 1 I
K
m
max max i
= + +
V V 1
1 I K
S K 1 I
K S
max
i
m
i
= + ⋅
+
+
Vmax/2 Vmaxi/2
Vmax Vmaxi
I
V V
1 I K
maxi max
i
= +
K K
1 I K
mi m
i
= +
Km V
S
I
nem inhibeált
1/S 1/V
I
I=0
Km/Vmax
1/Vmax
1 1
V
I K
i V
i
max max
= +
−1+ I K K
i m
1/Km
Uncompetitive inhibition
25
Linear mixed type inhibition
Mechanism of linear mixed type inhibition resembles to non- competitive inhibition but presence of I modifies the enzyme affinity to substrate.
Linear mixed type inhibition
Expressing the change of two kinetic parameters:
27
competitive noncompetitive uncompetitive
mixed
Summary of the inhibition types
S and I mutually exclude each other from the enzyme COMPETITIVE
S and I bind to the enzyme independently on each other NONCOMPETITIVE
I binds only after S UNCOMPETITIVE Like former but I modifies the affinity of the enzyme
MIXED TYPE
29
Substrate inhibition
The substrate binds to two or more sites.
If the S concentration is high, it can occur that two S bind to one and the other binding site forming inactive comp- lex.
(also reversible inhibition).
E
-OOC
CH2 CH2
-OOC
E
-OOC
CH CH
-OOC
Succinate Malonate
E
-OOCCH2CH2COO-
-OOCCH2CH2COO-
Normal S inhibition
Substrate inhibition
31 V
0.0 0.1 0.2 0.3 0.4
0 50 100 150 200 250 300
Vmax=0.9,Ks=50,Ki=10 Vmax=0.9,Ks=50,Ki=50
Vmax=0.9,Ks=50,Ki=100
Szubstrát koncentráció(mg/L)
