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Biochemistry 2/e - Garrett & Grisham
Chapter 16
Mechanisms of Enzyme Action
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
Outline
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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
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
16.2 Large Rate Accelerations
See Table 16.1
• Mechanisms of catalysis:
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– Entropy loss in ES formation
– Destabilization of ES
– Covalent catalysis
– General acid/base catalysis
– Metal ion catalysis
– Proximity and orientation
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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!
• Km cannot be "too tight" - goal is to make the
energy barrier between ES and EX‡ small
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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– a) loss of entropy due to formation of ES
– b) destabilization of ES by
• strain
• distortion
• desolvation
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
16.5 Transition State Analogs
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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!
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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The Serine Proteases
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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!
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Serine Protease Mechanism
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
The Aspartic Proteases
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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 OHin the transition state
• Other Asp (general acid) donates a proton,
facilitating formation of tetrahedral
intermediate
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Asp Protease Mechanism - II
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See 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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
HIV-1 Protease
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Therapy for HIV?
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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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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)
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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)
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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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