Enzymes Biological Catalysts

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Enzymes - Biological Catalysts (with
chymotrypsin as an example)
(chapter 6 of text)
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Example of a useful biochemical reaction
Peptide bond hydrolysis.
peptide hydrolysis reaction
This reaction takes years without a catalyst.
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Some ways to increase reaction rates of slow
reactions 1) increase temperature (not
very practical for living things) 2) use
enzyme as a catalyst to lower the activation
energy of a reaction
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Chymotrypsin catalyzes peptide bond hydrolysis.
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Some classes of enzymes
Protease - cleaves peptide bonds (example
chymotrypsin). Transferase - transfers
functional group. Isomerase - convert one
isomer to another. Ligase - Joining of 2
molecules together. Example DNA ligase
(requires ATP) etc.
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Reaction coordinate diagrams
Transition state
How do enzymes increase reaction rates?
By lowering the free energy of the transition
state.
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Enzymes contain active sites where substrates
bind and the chemical reaction takes place.
An enzyme can raise the local concentration of
reactants (by binding them close together), and
orient them most effectively for a reaction to
occur.
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Chymotrypsin - catalyzes peptide bond hydrolysis.
His
Asp
Ser
Active site of chymotrypsin is in red.
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Look at the Histidine Serine, with a peptide
substrate bound
peptide to be cleaved
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Histidine extracts proton from serine
Why does this happen? Why would His pull a
proton from Ser? What is the normal pKa of His
and Ser?
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Histidine extracts proton from serine
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Serine attacks peptide
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Tetrahedral intermediate
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Tetrahedral intermediate extracts proton from
histidine
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Cleaved peptide 1 is free to leave.
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Cleaved peptide 1 leaves, and is replaced by
water.
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Histidine extracts proton from water, OH- attacks
peptide bound to serine.
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Peptide 2 is free to leave.
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The catalytic mechanism of chymotrypsin and other
serine proteases
Figure 6.09
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The catalytic triad, with hydrogen bonding.
His
Asp
Ser
What does the Asp do? (stabilizes the histidine)
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So how does chymotrypsin speed up the reaction?
By stabilizing the transition state.
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The presence of the oxyanion hole helps to form
and stabilize the tetrahedral immediate. Activat
ion energy is much lower this way, compared to
simply breaking the peptide bond.
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Chymotrypsin will not cleave just any peptide
bond.
It has a preference cleaving after Phe or Tyr
(these a.a. fit in chymotrypsins substrate
binding pocket).
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Trypsin
Chymotrypsin
Elastase
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Divergent Evolution of Serine Proteases
3-D structures of chymotrypsin, trypsin, and
elastase
About 50 amino acid sequence similarity between
the 3 enzymes.
Figure 6.16
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Convergent evolution Subtilisin another
serine protease
lt15 sequence identity to trypsin, chymotrypsin,
or elastase i.e. not related
Figure 6.17 Structure of subtilisin from Bacillus
amyloliquefaciens.
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Thrombin is another well known serine
protease. Involved in blood coagulation. Specifi
c for a 4 a.a. sequence (compare with
chymotrypsin).
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Many more enzymes coming soon!
Hexokinase catalyzes the 1st step in glycolysis.
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Glycolysis (coming soon). Each step of 10 steps
is catalyzed by its own enzyme. Generates ATP.
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Chymotrypsin is made by ribosome in an inactive
form, secreted from cells. Must be cleaved to
fully activate. (chymotrypsin is a zymogen)
Why is chymotrypsin made in an inactive form?
(cant have an active protease loose inside cells)
Figure 6.19 Activation of chymotrypsinogen.
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Protease inhibitors - May look like substrates,
compete with natural substrate for active site.
Ritonavir is a protease inhibitor (Abbott
laboratories). Was supposed to inhibit HIV
protease. Now believed to be that it works by
inhibiting other proteases that degrade other HIV
inhibitors, allowing other HIV protease
inhibitors to persist longer in body. Ritonivir
is now used in low doses as part of a multi-drug
cocktail. 200 million to develop (mostly for
clinical trials).
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Chymotrypsin is an example of an acid-base
catalyst. Whats that? Mechanism involves a.a.
acting as acids and bases.
In chymotrypsin mechanism, serine donates proton,
His accepts proton.
So serine is acid, histidine is base.
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Would a simple mixture of Histidine Serine
catalyze peptide bond hydrolysis?
No - The His Ser must be in the context of the
enzyme to work.
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Amino acid side chains that can act as acid-base
catalysts
What do these have in common? What side chains
are not good H donors/acceptors?
Figure 6.05.
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Also, covalent catalysts
During the mechanism, an intermediate forms a
covalent link to the enzyme.
Tetrahedral intermediate
Chymotrypsin is also a covalent catalyst.
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Metal ion catalysts use a metal ion bound in
their active site.