Inhibited Enzyme Kinetics

Inhibitors may bind to enzyme and reduce

their activity.
Enzyme inhibition may be reversible or
irreversible.
For reversible enzyme inhibition, there are
- competitive
-noncompetitive
- uncompetitive
 Inhibited Enzyme Kinetics
Competitive inhibitors (I)
- substrate analogs
- compete with substrate for the active
site of the enzyme
K1
k2
E+S ES P E
K-1
+
I
KI
EI
 Inhibited Enzyme Kinetics
Competitive inhibitors (I)
Assume rapid equilibrium and with the
definitions of
d [ P]
v k [ ES ]
dt 2

' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ E ][ I ] [E0 ] [E] [ES ] [EI ]
KI
[ EI ]
 Inhibited Enzyme Kinetics
Competitive inhibitors (I),
we can obtain,
Vm [ S ]
v
' (1 [ I ] / K ) [ S ]
Km I
Vm [ S ]
v
'
Km , app [ S ]
' ' (1 [ I ] / K ) ' '
Km , app K m I When [I] =0, Km , app K m
Vm Remains that of Michaelis-Menten equation.
K m,app is an indication of the net effect of competitive
inhibition, ????? .
 Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
- not substrate analogs
- bind on site other than active sites

Km
k2
E+S ES P E
+ +
I I
KI KI
Km
EI+S ESI
 Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
Assume:
- rapid equilibrium
- same equilibrium constants of inhibitor
binding to E and ES KI
- same equilibrium constants of substrate
binding to E and EI Km
 Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
d [ P]
v k [ ES ]
dt 2

' k 1 [ E ][ S ] [ EI ][ S ]
Km
k1 [ ES ] [ ESI ]
[ E ][ I ] [ ES ][ I ]
KI
[ EI ] [ ESI ]

[E0 ] [E] [ES ] [EI ] [ESI ]

 Inhibited Enzyme Kinetics
Noncompetitive inhibitors (I)
we can obtain,
Vm [ S ]
v
' [ S ])
(1 [ I ] / K I )( K m
Vm, app[ S ]
v
' [S ]
Km
Km remains in Michaelis-Menten equation.
Vm,app is .
Vm, app Vm /(1 [ I ] / K I ) When [I] =0, Vm, app Vm
 Inhibited Enzyme Kinetics
Uncompetitive inhibitors (I)
- have no affinity for the enzyme itself
- bind only to the ES complex .
Assume rapid equilibrium,
Km
k2
E+S ES P E
+
I
KI
ESI
 Inhibited Enzyme Kinetics
Uncompetitive inhibitors (I)
d [ P]
v k [ ES ]
dt 2

' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ ES ][ I ]
KI
[ ESI ]

[E0 ] [E] [ES ] [ESI ]

 Inhibited Enzyme Kinetics
Uncompetitive inhibitors (I)
we can obtain,
(Vm /(1 [ I ] / K I ))[ S ]
v
' /(1 [ I ] / K )) [ S ]
(K m I
Vm, app[ S ]
v
' , app [ S ]
Km

Vm, app Vm /(1 [ I ] / K I ) '

Km K ' /(1 [ I ] / K )
, app m I
' '
K m, app / Vm, app K m / Vm - in Lineweaver-
Burk Plot
 Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors
- can cause inhibition at substrate
concentration
- bind only to the .
Assume rapid equilibrium,
Km
k2
E+S ES P E
+
S
K SI
ES2
Substrate Inhibition
 Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
d [ P]
v k [ ES ]
dt 2

' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ ES ][ S ]
K SI
[ ES 2]

[E0 ] [E] [ES ] [ES 2]

 Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
we can obtain,
Vm [ S ]
v
' [ S ] [ S ]2 / K
Km SI
At low substrate concentration [ S ]2 / K SI <<1
Vm [ S ]
v Michaelis-Menten Equation
Km' [S ]
'
At high substrate concentration K m /[S ] 1
Vm
v
1 [ S ] / K SI
 Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)

The substrate concentration resulting in the

maximum reaction rate can be determined by
setting dv/d[S]=0, [S]max is given by

Vm [S ]
dv / d [S ] d ( ) / d[S ] 0
' [ S ] [ S ]2 / K
Km SI

' K )1/ 2
[S ]max ( K m SI
 Uncompetitive substrate inhibitors (I)
Determine [S]max:
Vm [ S ]
dv / d [ S ] d ( ) / d[S ] 0
' [ S ] [ S ]2 / K
Km SI

Vm [ S ](1 2[ S ] / K SI )
Vm
( )0
' [ S ] [ S ]2 / K
Km ( K ' [ S ] [ S ]2 / K ) 2
SI m SI
' [ S ] [ S ]2 / K ) V [ S ](1 2[ S ] / K )
Vm ( K m SI m SI
( )0
(K m' [ S ] [ S ]2 / K ) 2
SI
' V [ S ]2 / K
Vm K m m SI
)0
' [ S ] [ S ]2 / K ) 2
(K m SI
' V [ S ]2 / K 0
Vm K m m SI
' K )1 / 2
[ S ]max ( K m SI
 Inhibition Estimation
Product formation rate v ~ [S]: v has a peak?
If yes, then its substrate inhibition.
- get [S]max from the plot of v~[s].
- at low substrate concentration, obtain Vm and Km
graphically or through direct calculation.
- calculate KI through
' K )1/ 2
[S ]max ( K m SI

If no, then examine the data with and without inhibitors in

1/v ~ 1/[S] plot (Lineweaver-Burk Plot).
Estimation of Inhibited Enzyme
Kinetics
 Estimation of Inhibited Enzyme
Kinetics
Determine the type of inhibition.
Determine the parameters for Michaelis-
Menten equation without inhibition.
Determine the parameter of KI for inhibited
kinetics.
 Summary of Inhibited Kinetics
For reversible enzyme inhibition, there are
- competitive
- noncompetitive
- uncompetitive
- substrate inhibition

Determine parameters for all these types

of inhibition kinetics.