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Chapter 16

Chapter 16. Mechanisms of Enzyme Action. All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777. Outline.

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Chapter 16

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  1. Chapter 16 Mechanisms of Enzyme Action All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 16.1 Stabilization of the Transition State • 16.2 Enormous Rate Accelerations • 16.3 Binding Energy of ES • 16.4 Entropy Loss and Destabilization of ES • 16.5 Transition States Bind Tightly • 16.6 - 16.9 Types of Catalysis • 16.11 Serine Proteases • 16.12 Aspartic Proteases • 16.13 Lysozyme (probably will not cover)

  3. 16.1 Stabilization of the Transition State • Rate acceleration (or enhancement) by an enzyme means that the energy barrier between ES and EX‡ must be smaller than the barrier between S and X‡ • This means that the enzyme must stabilize the EX‡ transition state more than it stabilizes ES

  4. 16.2 Rate Accelerations • Mechanisms of catalysis: • Entropy loss in ES formation • Destabilization of ES • Covalent catalysis • General acid/base catalysis • Metal ion catalysis • Proximity and orientation

  5. 16.3 Binding Energy of ES Competing effects determine the position of ES on the energy scale • What are the favorable and unfavorable binding effects at the catalytic site ??? • The binding of S to E must be favorable, but not too favorable! • Km cannot be too small (i.e., "too tight”) - goal is for the energy barrier between ES and EX‡ to be small!

  6. 16.4 Entropy Loss and Destabilization of the ES Complex Raising the energy of ES actually increases the rate! WHY? • For a given energy of EX‡, raising the energy of ES will increase the enzyme catalyzed rate • This is accomplished by • a) loss of entropy due to formation of ES • translational and rotational • b) destabilization of ES by • strain • distortion • desolvation

  7. 16.5 Transition State Analogs Very tight binding to the enzyme catalytic site! • The affinity of the enzyme for the transition state may be quite strong! • Is it possible to achieve such binding affinities with stable molecules? • Transition state analogs come close! • Proline racemase was the first example

  8. 16.6 Covalent Catalysis Serine Proteases provide examples! • Enzyme and substrate become linked by a covalent bond at one or more points in the reaction pathway

  9. Covalent Catalysis • For example, consider the following SN2 reaction: Nu: - + R-L ----> Nu-R + :L- • If in the enzyme catalyzed reaction: E + R-L ---> E-R + :L- (and then) E-R + Nu: - ---> E + R-L • There will be rate enhancement if the functional group in the enzyme catalytic site that reacts with R-L is a better nucleophile than Nu and a better leaving group than L

  10. General Acid-base Catalysis Catalysis in which a proton is transferred in the transition state • "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalytic center • "General" acid-base catalysis involves acids and bases other than H+ and OH- • These other acids and bases facilitate transfer of H+ in the transition state

  11. Proximity • Chemical reactions are accelerated by having the reactants in close proximity - that is near each other • We saw an example of this effect with PNP!

  12. The Serine Proteases Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA (tissue plasminogen activator) • All utilize a serine residue in the catalyzed reaction • Ser is part of a "catalytic triad" of Ser, His, Asp • Serine proteases are homologous, but locations of the three crucial residues differ somewhat • Enzymologists agree, however, to number them always as His-57, Asp-102, Ser-195 • Burst kinetics yields a hint of how they work!

  13. Mechanism for Serine Proteases Like Chymotrypsin and Trypsin A mixture of covalent and general acid-base catalysis • Asp-102 functions primarily to orient His-57 • His-57 acts as a general acid and base • Ser-195 forms a covalent bond with peptide to be cleaved • Covalent bond formation turns a trigonal C into a tetrahedral C • The tetrahedral intermediate is stabilized by N-Hs of Gly-193 and Ser-195

  14. Artificial Substrates • P-Nitrophenyl acetate • Acetylphenylalanine methyl ester • Benzolyalanine methyl ester

  15. The Aspartic Proteases Pepsin, chymosin, cathepsin D, renin and HIV-1 protease • All involve two Asp residues at the active site • Two Asps work together as general acid-base catalysts • HIV-1 protease is an axample and is a homodimer

  16. Aspartic Protease Mechanism The pKa values of the Asp residues are crucial • One Asp has a relatively low pKa, other has a relatively high pKa • Deprotonated Asp acts as general base, accepting a proton from HOH, forming OH- in the transition state • Other Asp (general acid) donates a proton, facilitating formation of tetrahedral intermediate

  17. HIV-1 Protease A novel aspartic protease • HIV-1 protease cleaves the polyprotein products of the HIV genome • This is a remarkable imitation of mammalian aspartic proteases • HIV-1 protease is a homodimer - more genetically economical for the virus • Active site is two-fold symmetric • Two Asp residues - one high pKa, one low pKa

  18. Therapy for HIV? Protease inhibitors as AIDS drugs • If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form • Several novel protease inhibitors are currently marketed as AIDS drugs • Many such inhibitors work in a culture dish • However, a successful drug must be able to kill the virus in a human subject without blocking other essential proteases in the body

  19. Lysozyme • Lysozyme hydrolyzes polysaccharide chains and ruptures certain bacterial cells by breaking down the cell wall • Hen egg white enzyme has 129 residues with four disulfide bonds • The first enzyme whose structure was solved by X-ray crystallography (by David Phillips in 1965)

  20. Substrate Analog Studies • Natural substrates are not stable in the active site for structural studies • But analogs can be used - like (NAG)3 • Fitting a NAG into the D site requires a distortion of the sugar • This argues for stabilization of a transition state via destabilization (distortion and strain) of the substrate

  21. The Lysozyme Mechanism • Studies with 18O-enriched water show that the C1-O bond is cleaved on the substrate between the D and E sites • This incorporates 18O into C1 • Glu35 acts as a general acid • Asp52 stabilizes a carbocation intermediate (see Figure 16.37)

  22. Chapter 16 Problems • Note in the Science article referenced in number 2 that the figure legend has a mistake!

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