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 -

Inhibited Enzyme Kinetics

Competitive inhibitors (I) - substrate analogs - compete with substrate for the active site of the enzyme
K1

E+S
+

K-1

k2 ES P E

KI
EI

Inhibited Enzyme Kinetics

Competitive inhibitors (I) Assume rapid equilibrium and with the definitions of

d[P] v k [ES] 2 dt k 1 [ E ][S ] ' Km k1 [ ES ]

[ E ][I ] KI [ EI ]

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

Inhibited Enzyme Kinetics

Competitive inhibitors (I), we can obtain,
v Vm [ S ]

' (1 [ I ] / K ) [ S ] Km I

Vm [ S ] ' Km , app [ S ]

' ' (1 [ I ] / K ) Km K , app m I When [I] =0,

' ' Km K , app m

Vm Remains

that of Michaelis-Menten equation. .

K m ,app is

Inhibited Enzyme Kinetics

Noncompetitive inhibitors (I) - not substrate analogs - bind on
Km

E+S
+

k2 ES P E
+

I KI
Km

I
KI ESI

EI+S

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 Km binding to E and EI

Inhibited Enzyme Kinetics

Noncompetitive inhibitors (I)

d[P] v k [ES] 2 dt 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,
v Vm [ S ] ' [ S ]) (1 [ I ] / K I )(K m

Vm, app [ S ] v ' [S ] Km

K m 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 - bind only to the Assume rapid equilibrium,
Km

itself
.

E+S

k2 ES P E
+

KI ESI

Inhibited Enzyme Kinetics

Uncompetitive inhibitors (I)

d[P] v k [ES] 2 dt k 1 [ E ][S ] ' Km k1 [ ES ]

[ ES ][I ] KI [ ESI ]

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

Inhibited Enzyme Kinetics

Uncompetitive inhibitors (I) we can obtain,
v (Vm /(1 [ I ] / K I ))[S ] ' /(1 [ I ] / K )) [ S ] (K m I

Vm, app [ S ] v ' , app [ S ] Km ' ' /(1 [ I ] / K ) Vm, app Vm /(1 [ I ] / K I ) K m K , app m I

' ' K m, app / Vm, app K m / Vm Burk Plot

in Lineweaver-

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
+

K SI ES2

Substrate Inhibition

Inhibited Enzyme Kinetics

Uncompetitive substrate inhibitors (I)

d[P] v k [ES] 2 dt k 1 [ E ][S ] ' Km k1 [ ES ]

[ ES ][S ] K SI [ ES 2]

[E0] [E] [ES] [ES2]

Inhibited Enzyme Kinetics

Uncompetitive substrate inhibitors (I) we can obtain,
v Vm [S ] ' [ S ] [S ]2 / K Km SI

At low substrate concentration [S ]2 / K <<1 SI

' At high substrate concentration K m /[ S ] 1

Vm [ S ] v ' [S ] Km
v Vm

Michaelis-Menten Equation

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

' K )1/ 2 [S ]max (Km SI

 Uncompetitive substrate inhibitors (I) Determine [S]max:

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

Vm [ S ](1 2[ S ] / K SI ) ( )0 ' [ S ] [ S ]2 / K ' [ S ] [ S ]2 / K ) 2 Km ( K m SI SI Vm ' [ S ] [ S ]2 / K ) V [ S ](1 2[ S ] / K ) Vm ( K m m SI SI ( )0 ' [ S ] [ S ]2 / K ) 2 (K m SI ' V [ S ]2 / K Vm K m m SI ' [ S ] [ S ]2 / K ) 2 (K m SI )0

' V [ S ]2 / K 0 Vm K m m SI ' K )1 / 2 [ S ]m ax ( 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 (Km 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 MichaelisMenten 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.