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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 16 Mechanisms of Enzyme Action to accompany Biochemistry, 2/e.

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Presentation on theme: "Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 16 Mechanisms of Enzyme Action to accompany Biochemistry, 2/e."— Presentation transcript:

1 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 16 Mechanisms of Enzyme Action to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham 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

2 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 13.1 Stabilization of the Transition State 13.2 Enormous Rate Accelerations 13.3 Binding Energy of ES 13.4 Entropy Loss and Destabilization of ES 13.5 Transition States Bind Tightly Types of Catalysis Serine Proteases Aspartic Proteases Lysozyme

3 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.1 Stabilizing the Transition State Rate acceleration 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 See Eq. 16.3

4 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

5 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.2 Large Rate Accelerations See Table 16.1 Mechanisms of catalysis: –Entropy loss in ES formation –Destabilization of ES –Covalent catalysis –General acid/base catalysis –Metal ion catalysis –Proximity and orientation

6 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.3 Binding Energy of ES Competing effects determine the position of ES on the energy scale Try to mentally decompose the binding effects at the active site into favorable and unfavorable The binding of S to E must be favorable But not too favorable! K m cannot be "too tight" - goal is to make the energy barrier between ES and EX ‡ small

7 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

8 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

9 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.4 Entropy Loss and Destabilization of ES Raising the energy of ES raises the rate For a given energy of EX ‡, raising the energy of ES will increase the catalyzed rate This is accomplished by –a) loss of entropy due to formation of ES –b) destabilization of ES by strain distortion desolvation

10 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

11 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

12 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

13 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.5 Transition State Analogs Very tight binding to the active site! The affinity of the enzyme for the transition state may be M! Can we see anything like that with stable molecules? Transition state analogs (TSAs) do pretty well! Proline racemase was the first case See Figure 16.8 for some good recent cases!

14 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

15 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

16 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 16.6 Covalent Catalysis Serine Proteases are good examples! Enzyme and substrate become linked in a covalent bond at one or more points in the reaction pathway The formation of the covalent bond provides chemistry that speeds the reaction

17 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

18 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

19 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 See Figure 16.12

20 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

21 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

22 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 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 yield a hint of how they work!

23 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

24 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Serine Protease Mechanism A mixture of covalent and general acid-base catalysis Asp-102 functions only 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 oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195

25 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

26 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

27 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

28 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

29 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

30 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

31 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

32 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

33 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

34 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 Most aspartic proteases have a tertiary structure consisting of two lobes (N-terminal and C-terminal) with approximate two-fold symmetry HIV-1 protease is a homodimer

35 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

36 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Aspartic Protease Mechanism The pK a values of the Asp residues are crucial One Asp has a relatively low pK a, other has a relatively high pK a 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

37 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

38 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Asp Protease Mechanism - II See Figure What evidence exists to support the hypothesis of different pK a values for the two Asp residues? See the box on page 525 Bell-shaped curve is a summation of the curves for the two Asp titrations In pepsin, one Asp has pK a of 1.4, the other 4.3

39 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

40 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

41 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 pK a, one low pK a

42 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

43 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

44 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

45 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

46 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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)

47 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

48 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

49 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

50 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

51 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

52 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

53 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Lysozyme Mechanism Studies with 18 O-enriched water show that the C 1 -O bond is cleaved on the substrate between the D and E sites This incorporates 18 O into C 1 Glu 35 acts as a general acid Asp 52 stabilizes a carbonium ion intermediate (see Figure 16.37)

54 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

55 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

56 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 16 Problems Work the end-of-chapter problems! Number 2 is particularly good Note in the Science article referenced in number 2 that the figure legend has a mistake!


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