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Chapter 6

Chapter 6. Mechanisms of Enzyme Action. 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)

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Chapter 6

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  1. 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

  4. 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)

  5. 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.

  6. 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.

  7. 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

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

  9. Mechanistic Strategies

  10. Polar AA Residues in Active Sites

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

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

  13. 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)

  14. 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

  15. 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:-)

  16. : : Acid-Base Catalysis carbanion intermediate

  17. 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)

  18. 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-

  19. 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

  20. Serine Proteases are structurally Similar Chymotrpsin Trypsin Elastase

  21. Substrate binding specificity

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