E nzyme inhibition Irreversible and reversible inhibition

1. Enzyme inhibition Irreversible and reversible inhibition * Irreversible: inhibitor becomes covalently bound to enzyme thereby diminishing its activity * Can be used for the elucidation of functional groups at the enzyme active site * group-specific reagents affinity labels suicide inhibitors

2. Group-specific reagents React with specific R-groups of amino acids * Diisopropylphosphofluoridate Inhibits an enzyme by covalently modifying a crucial serine residue * Iodoacetamide Inactivates an enzyme by reacting with a critical cysteine residue

5. Affinity labels Structurally similar to the enzyme substrate More specific than group-specific reagents * Tosyl-L-phenylalanine chloromethylketone Substrate analog for chymotrypsin Inactivates chymotrypsin by reacting with an essential histidine residue

7. Suicide inhibitors Mechanism-based inhibitors * Modified substrates that provide the most specific means to modify an enzyme active site Initially processed by the normal catalytic mechanism Generates a chemically reactive intermediate that inactivates the enzyme through covalent modification

8. Suicide inhibitors The enzyme participates in its own irreversible inhibition Strongly suggests that the covalently modified group on the enzyme is catalytically vital Monoamine oxidase (MAO) inhibition by N,N-dimethylpropargylamine and (S-)deprenyl Analogs of dopamine and serotonin Treatment of Parkinson disease

11. Reversible inhibition Rapid dissociation of the enzyme-inhibitor complex * Competitiveinhibition * Non-competitiveinhibition * Mixed-typeinhibition Kinetic experiments can provide information on the mechanism of inhibition and, thus, on the structural characteristics of the enzyme active site

12. Competitive inhibition Enzyme can bind substrate (forming an ES complex) or inhibitor (EI) but not both (ESI) * Competitive inhibitor resembles the substrate * Competive inhibition canbe relieved by increasing the substrate concentration

13. Dihydrofolate reductase Involved in the biosynthesis of purines and pyrimidines *Methotrexateis a structural analog of tetrahydrofolate, a coenzyme for DHFR * Methotrexate binds to DHFR 1000-fold more tightly than tetrahydrofolate and inhibits nucleotide base synthesis * Used to treat cancer

15. Non-competitive inhibition Inhibitor and substratebind simultaneously to an enzyme molecule at different binding sites (ESI) * Non-competitive inhibitor acts by decreasing theturnover number rather than by diminishing the proportion of enzyme molecules that are bound by substrate * Non-competive inhibition cannot be overcome by increasing the substrate concentration

17. Competitive inhibition Inhibitor competes with the substrate at the substrate binding site Vmax remains the same

19. Non-competitive inhibition Substrate can still bind to the enzyme-inhibitor complex Vmax is decreased

21. Analysis of reversible inhibition Non-linear regression analysis * Lineweaver-Burkplots Double reciprocal plots are especially useful for distinguishing between competitive and non-competitive inhibitors

22. Lineweaver Burk plot

26. Substrate inhibition With some enzymes high substrate concentrations cause enzyme inhibition * Too much of a good thing Binding of the substrate at a second site results in “non-competitive” inhibition

27. Substrate inhibition

28. Inhibition by transition-state analogs Proline racemase Planar transition state,trigonal negatively charged -carbon atom Pyrrole 2-carboxylatebinds 160-fold more tightly than L-proline Inhibitory power of transition-state analogs underscores the essence of catalysis: selective binding of the transition state

30. Transition-state analogs as irreversible inhibitors Penicillin Blocks last step in cell-wall synthesis, i.e. the cross-linking of peptidoglycan strands Glycopeptide transpeptidase Trojan horsestrategem