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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chapter 16
Mechanisms of Enzyme Actionto 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 32887-6777
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
• 13.6 - 13.9 Types of Catalysis
• 13.11 Serine Proteases
• 13.12 Aspartic Proteases
• 13.13 Lysozyme
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
16.3 Binding Energy of ESCompeting 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!
• Km cannot be "too tight" - goal is to make the energy barrier between ES and EX‡ small
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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 10 -15 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!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
General Acid-base CatalysisCatalysis 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The Serine ProteasesTrypsin, 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!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Serine Protease MechanismA 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Aspartic Protease MechanismThe 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Asp Protease Mechanism - IISee Figure 16.27
• What evidence exists to support the hypothesis of different pKa 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 pKa of 1.4, the other 4.3
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
HIV-1 ProteaseA 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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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)
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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 carbonium ion intermediate (see Figure 16.37)
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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!