1. Mechanisms of Enzyme Action
Chapter 6
Mechanisms of Enzyme Action

2. Enzymatic Catalysis
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 transition state (TS)
The higher [TS], the move likely the rxn will proceed.

3. Enzymatic Catalysis
S  Ts  P

5. 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 secs)

6. 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.

7. Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation, decrease entropy)
Need to stabilize TS to lower activation energy barrier.

8. 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

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

10. Mechanistic Strategies

11. Polar AA Residues in Active Sites

13. Common types of enzymatic mechanisms
Substitutions rxns
Bond cleavage rxns
Redox rxns
Acid base catalysis
Covalent catalysis

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

15. 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)

16. Cleavage Rxns Heterolytic vs homolytic cleavage
Carbanion formation (retains both e-) R3-C-H  R3-C:- + H+
Carbocation formation (lose both e-) R3-C-H  R3-C+ + H:-
Free radical formation (lose single e-) R1-O-O-R2  R1-O* + *O-R2
Hydride ion

17. Acid-Base Catalysis Accelerates rxn by catalytic transfer of a proton
Involves AA residues that can accept a proton
Can remove proton from –OH, -NH, -CH, or –XH
Creates a strong nucleophillic reactant (i.e. X:-)

18. Acid-Base Catalysis :
carbanion intermediate

19. 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)

20. Covalent Catalysis Protein Kinases
ATP + E + Protein <-> ADP + E + Protein-P
A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4-
E-O-PO4- + Protein-OH <-> E + Protein-O- PO4-

21. Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA
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

23. Serine Proteases are structurally Similar
Chymotrpsin Trypsin Elastase

25. Substrate binding specificity