1. Mechanisms of Enzyme Action

2. Transition (TS) State Intermediate Transition state = unstable high-energy intermediate Rate of rxn depends on the frequency at which reactants collide and form the TS Reactants must be in the correct orientation and collide with sufficient energy to form TS Bonds are in the process of being formed and broken in TS Short lived (10 –14 to 10 -13 secs)

3. Activation Energy (AE) – The energy require to reach transition state from ground state. AE barrier must be exceeded for rxn to proceed. Lower AE barrier, the more stable the TS The higher [TS], the move likely the rxn will proceed. Transition State (TS) Intermediate

4. Intermediates Intermediates are stable. In rxns w/ intermediates, 2 TS’s are involved. The slowest step (rate determining) has the highest AE barrier. Formation of intermediate is the slowest step.

5. Catalyst stabilize the Transition State Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation) Need to stabilize TS to lower activation energy barrier.

6. Common types of enzymatic mechanisms Substitutions rxns Bond cleavage rxns Redox rxns

7. Substitution Rxns Nucleophillic Substitution– Direct Substitution transition state Nucleophillic = e - rich Electrophillic = e - poor

8. Heterolytic vs homolytic cleavage Carbanion formation (retains both e - ) R 3 -C-H  R 3 -C: - + H + Carbocation formation (lose both e - ) R 3 -C-H  R 3 -C + + H: - Free radical formation (lose single e - ) R 1 -O-O-R 2  R 1 -O* + *O-R 2 Cleavage Rxns Hydride ion

9. Oxidation reduction (Redox) Rxns Loose e - = oxidation (LEO) Gain e - = reduction (GER) Central to energy production If something oxidized something must be reduced (reducing agent donates e - to oxidizing agent) Oxidations = removal of hydrogen or addition of oxygen or removal of e - In biological systems reducing agent is usually a co-factor (NADH of NADPH)

10. Polar AA Residues in Active Sites ReactiveCharge AAGroup@pH 7Functions Aspartate-COO - -1Cation Binding, H + transfer Glutamate-COO - -1Cation Binding, H + transfer HistidineImidazole~0H + transfer Cysteine-CH 2 SH~0Binding of acyl groups TyrosinePhenol 0H-Bonding to ligands Lysine-NH 3 + +1Anion binding, H + transfer ArginineGuanidinium+1Anion binding Serine -CH 2 OH 0Binding of acyl groups

11. Accelerates rxn by catalytic transfer of a proton Involves AA residues that can donate or transfer protons at neutral pH (e.g. histidine) HB + :B + H + Acid-Base Catalysis

12. : : carbanion intermediate

13. Covalent Catalysis 20% of all enzymes employ covalent catalysis A-X + B + E BX + E + A A group from a substrate binds covalently to enzyme (A-X + E A + X-E) The intermediate enzyme substrate complex (A-X) then donates the group (X) to a second substrate (B) (B + X-E B-X + E)

14. Covalent Catalysis Protein Kinases ATP + E + Protein ADP + E + Protein-P 1)A-P-P-P(ATP) + E-OH A-P-P (ADP) + E-O-PO 4 - 2)E-O-PO 4 - + Protein-OH E + Protein-O- PO 4 -

15. Binding Modes of Enzymatic Catalysis Proximity Effects Transition State Stabilization

16. Proximity Effects In multi-substrate reactions, collecting and correct positioning of substrates important Increases effective concentration of substrates which favors formation of TS Decreases activation energy by decreasing entropy Increases rate of reaction > 10,000 fold E binding to S can not be too strong or conversion from ES to TS would require too much energy

17. ES complex must not be too stable Raising the energy of ES will increase the catalyzed rate This is accomplished by loss of entropy due to formation of ES and destabilization of ES by strain distortion desolvation

21. ES complex must not be too stable Raising the energy of ES will increase the catalyzed rate This is accomplished by loss of entropy due to formation of ES and destabilization of ES by strain distortion desolvation

22. Transition State Stabilization Transition state analog Equilibrium between ES TS, enzyme drives equilibrium towards TS Enzyme binds more tightly to TS than substrate

23. The Serine Proteases Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA All involve a serine in catalysis - thus the name Ser is part of a "catalytic triad" of Ser, His, Asp (show over head) Serine proteases are homologous, but locations of the three crucial residues differ somewhat Substrate specificity determined by binding pocket

24. Serine Proteases are structurally Similar

25. Substrate binding specificity