Inhibited Enzyme Kinetics

1. 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

2. Inhibited Enzyme Kinetics • Competitive inhibitors (I) - substrate analogs - compete with substrate for the active site of the enzyme K1 E+S K-1 + I K I EI

3. Inhibited Enzyme Kinetics • Competitive inhibitors (I) Assume rapid equilibrium and with the definitions of

4. Inhibited Enzyme Kinetics • Competitive inhibitors (I), we can obtain, When [I] =0, remains same as that in Michaelis-Menten equation. is increased.

5. Inhibited Enzyme Kinetics • Noncompetitive inhibitors (I) - not substrate analogs - bind on sites other than the active sites Km’ E+S + + I I K I K I Km’ ESI EI+S

6. 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’

7. Inhibited Enzyme Kinetics • Noncompetitive inhibitors (I)

8. Inhibited Enzyme Kinetics • Noncompetitive inhibitors (I) we can obtain, remains same as that in Michaelis-Menten equation. is decreased. When [I] =0,

9. Inhibited Enzyme Kinetics • Uncompetitive inhibitors (I) - have no affinity for the enzyme itself - bind only to the ES complex Assume rapid equilibrium, Km’ E+S + I K I ESI

10. Inhibited Enzyme Kinetics • Uncompetitive inhibitors (I)

11. Inhibited Enzyme Kinetics • Uncompetitive inhibitors (I) we can obtain, -same slopes in Lineweaver-Burk Plot

12. Inhibited Enzyme Kinetics • Uncompetitive substrate inhibitors - can cause inhibition at high substrate concentration - bind only to the ES complex Assume rapid equilibrium, Km’ E+S + S K SI ES2

13. Substrate Inhibition

14. Inhibited Enzyme Kinetics • Uncompetitive substrate inhibitors (I)

15. Inhibited Enzyme Kinetics • Uncompetitive substrate inhibitors (I) we can obtain, At low substrate concentration <<1 Michaelis-Menten Equation At high substrate concentration

16. 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

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

18. 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).

19. Estimation of Inhibited Enzyme Kinetics

20. 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.

21. Summary of Inhibited Kinetics • For reversible enzyme inhibition, there are - competitive - noncompetitive - uncompetitive - substrate inhibition • Determine parameters for all these types of inhibition kinetics.