lecture notes enzyme 3 inhibition kinetics web

21
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 - - -

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Page 1: Lecture Notes Enzyme 3 Inhibition Kinetics Web

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

-

-

-

Page 2: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Competitive inhibitors (I)

- substrate analogs

- compete with substrate for the active site of the enzyme

K1

EPk

ES 2E+SK-1

+

I

EI

K I

Page 3: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Competitive inhibitors (I)

Assume rapid equilibrium and with the definitions of

][][][]0[ EIESEE

][][

2ESk

dt

Pdv

][

]][[

1

1'ES

SE

k

kmK

][

]][[

EI

IEIK

Page 4: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics• Competitive inhibitors (I),

we can obtain,

][)/][1('

][

SIKImK

SmVv

][',

][

SappmK

SmVv

)/][1('', IKImKappmK When [I] =0, ''

, mKappmK

mV Remains that of Michaelis-Menten equation.

appmK , is .

Page 5: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Noncompetitive inhibitors (I)

- not substrate analogs

- bind on .

EPk

ES 2E+S+

I

EI+S

K I

+

I

ESI

K IKm’

Km’

Page 6: Lecture Notes Enzyme 3 Inhibition Kinetics Web

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’

Page 7: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Noncompetitive inhibitors (I)

][][][][]0[ ESIEIESEE

][][

2ESk

dt

Pdv

][

]][[

][

]][[

1

1'ESI

SEI

ES

SE

k

kmK

][

]][[

][

]][[

ESI

IES

EI

IEIK

Page 8: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics• Noncompetitive inhibitors (I)

we can obtain,

])[')(/][1(

][

SmKIKI

SmVv

]['

][,

SmK

SappmVv

)/][1/(, IKImVappmV When [I] =0, mVappmV ,

appmV ,

remains in Michaelis-Menten equation.mK

is .

Page 9: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Uncompetitive inhibitors (I)

- have no affinity for itself

- bind only to the .

Assume rapid equilibrium,

EPk

ES 2E+S+

I

ESI

K I

Km’

Page 10: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Uncompetitive inhibitors (I)

][][][]0[ ESIESEE

][][

2ESk

dt

Pdv

][

]][[

1

1'ES

SE

k

kmK

][

]][[

ESI

IESIK

Page 11: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics• Uncompetitive inhibitors (I)

we can obtain,

][))/][1/('(

]))[/][1/((

SIKImK

SIKImVv

][,'

][,

SappmK

SappmVv

)/][1/(, IKImVappmV )/][1/('', IKImKappmK

mVmKappmVappmK /',/', - in Lineweaver-

Burk Plot

Page 12: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Uncompetitive substrate inhibitors

- can cause inhibition at substrate concentration

- bind only to the .

Assume rapid equilibrium,

EPk

ES 2E+S+

S

ES2

K SI

Km’

Page 13: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Substrate Inhibition

Page 14: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics

• Uncompetitive substrate inhibitors (I)

][][][]0[ 2ESESEE

][][

2ESk

dt

Pdv

][

]][[

1

1'ES

SE

k

kmK

][

]][[

2ES

SESSIK

Page 15: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibited Enzyme Kinetics• Uncompetitive substrate inhibitors (I)

we can obtain,

SIKSSmK

SmVv/2][]['

][

]['][

SmK

SmVv

At low substrate concentration SIKS /2][ <<1

SIKS

mVv/][1

At high substrate concentration 1]/[' SmK

Michaelis-Menten Equation

Page 16: Lecture Notes Enzyme 3 Inhibition Kinetics Web

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

0][/)/2][]['

][(][/

Sd

SIKSSmK

SmVdSddv

2/1)'(max][ SIKmKS

Page 17: Lecture Notes Enzyme 3 Inhibition Kinetics Web

2/1)'(max][

0/2]['

0)2)/2][]['(

/2]['

0)2)/2][]['(

)/][21]([)/2][]['((

0)2)/2][]['(

)/][21]([(

/2][]['

0][/)/2][]['

][(][/

SIKmKS

SIKSmVmKmV

SIKSSmK

SIKSmVmKmV

SIKSSmK

SIKSSmVSIKSSmKmV

SIKSSmK

SIKSSmV

SIKSSmK

mV

Sd

SIKSSmK

SmVdSddv

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

Page 18: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Inhibition Estimation

• Product formation rate v ~ [S]: v has a peak?

If yes, then it’s 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

If no, then examine the data with and without inhibitors in 1/v ~ 1/[S] plot (Lineweaver-Burk Plot).

2/1)'(max][ SIKmKS

Page 19: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Estimation of Inhibited Enzyme Kinetics

Page 20: Lecture Notes Enzyme 3 Inhibition Kinetics Web

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.

Page 21: Lecture Notes Enzyme 3 Inhibition Kinetics Web

Summary of Inhibited Kinetics

• For reversible enzyme inhibition, there are

- competitive

- noncompetitive

- uncompetitive- substrate inhibition

• Determine parameters for all these types of inhibition kinetics.