Strategies in Enzyme Catalysis

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Strategies in Enzyme Catalysis

• As stated earlier, the role of a catalyst is to
  decrease the energy of activation of a
  reactionthe energy necessary to attain the
  transition state.
• Several themes recur in enzyme catalysis.
• Catalysis by approximation
• General acid, general base catalysis
• Catalysis by electrostatic effects
• Covalent catalyis (nucleophilic or electrophilic)
• Catalysis by strain or distortion
• For most enzymes, more than one of these
  strategies are used concomitantly

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Catalysis by Approximation

• The classic way that an enzyme increases the rate
  of a bimolecular reaction is to use binding
  energy to simply bring the two reactants in close
  proximity.
• If DG is the change in free energy between the
  ground state and the transition state, then
  DGDHtDS. In solution, the transition state
  would be significantly more ordered than the
  ground state, and DS would therefore be
  negative.
• The formation of a transition state is
  accompanied by losses in translational entropy as
  well as rotational entropy. Enzymatic reactions
  take place within the confines of the enzyme
  active-site wherein the substrate and catalytic
  groups on the enzyme act as one molecule.
  Therefore, there is no loss in translational or
  rotational energy in going to the transition
  state.
• This is paid for by binding energy.

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Entropy and Catalysis
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Orientation Effects
In the non-enzymatic lactonization reaction shown
below, the relative rate when R CH3 is 3.4
x1011 times that when R H. What is the
explanation?
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Catalysis by Approximation
In order for a reaction to take place between two
molecules, the molecules must first find each
other. This is why the rate of a reaction is
dependent upon the concentrations of the
reactants, since there is a higher probability
that two molecules will collide at high
concentrations. As an example, look at the
hydrolysis of paranitrophenyl ester again
catalyzed by imidazole. This reaction depends on
both the concentration of imidazole and
paranitrophenyl ester, therefore, it proceeds
with a Second Order Rate Constant of 35 M-1min-1.
In the second reaction, the imidazole catalyst
is actually part of the substrate that is being
hydrolyzed. Therefore, the rate of hydrolysis is
dependent only on the substrate, and therefore
proceeds with a First Order Rate Constant of 839
min-1. Rate constants of different order cannot
be compared. However, the ratio of the first
order rate constant to the second order rate
constant gives an effective Molarity. In order
for the second order reaction to be as fast as
the first order reaction, it would be necessary
to have imidazole at a concentration of 24 M!
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Effective Concentration
Effective concentration is k1/k2 2 x 105 M
Effective concentration 2 x 107 M
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General Acid-Base Catalysis

• General acid-base catalysis is involved in a
  majority of enzymatic reactions. General
  acidbase catalysis needs to be distinguished
  from specific acidbase catalysis.
• Specific acidbase catalysis means specifically,
  OH or H accelerates the reaction. The reaction
  rate is dependent on pH only, and not on buffer
  concentration.
• In General acidbase catalysis, the buffer aids
  in stabilizing the transition state via donation
  or removal of a proton. Therefore, the rate of
  the reaction is dependent on the buffer
  concentration, as well as the appropriate
  protonation state.

Specific base catalysis
General base catalysis
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General Base Catalysis and Ester Hydrolysis
In the second step (collapse of the tetrahedral
intermediate), the leaving group must be
protonated. The general acidbase is best when
its pKa is near that of the pH of the solution,
in order to have appropriate concentrations of
each buffer species.
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Hydrolysis of Paranitrophenylacetate
The hydrolysis of esters proceeds readily under
in the presence of hydroxide. It is base
catalyzed. However, the rate of hydrolysis is
also dependent on imidazole buffer concentration.
Imidazole can accept a proton from water in the
transiton state in order to generate the better
nucleophile, hydroxide. It can also re-donate
the proton to the paranitrophenylacetate in order
to generate a good leaving group.
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Conventions for Describing General Acid/Base
Catalysis
The dehydration reaction below is catalyzed by an
enzyme at pH 7 and 25C. This reaction does not
occur nonenzymatically under these conditions.
Sketch a mechanism to show how an enzyme can
easily catalyze this reaction.
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Dehydration Mechanism
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Electrostatic Effects
Electrostatic interactions are much stronger in
organic solvents than in water due to the
dielectric constant of the medium. The interior
of enzymes have dielectric constants that are
similar to hexane or chloroform
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Catalysis by Metal Ions-1
Metal ions that are bound to the protein
(prosthetic groups or cofactors) can also aid in
catalysis. In this case, Zinc is acting as a
Lewis acid. It coordinates to the non-bonding
electrons of the carbonyl, inducing charge
separation, and making the carbon more
electrophilic, or more susceptible to
nucleophilic attack.
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Catalysis by Metal Ions-2
Metal ions can also function to make potential
nucleophiles (such as water) more nucleophilic.
For example, the pka of water drops from 15.7 to
6-7 when it is coordinated to Zinc or Cobalt.
The hydroxide ion is 4 orders of magnitude more
nucleophilic than is water.
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Covalent Catalysis
There must be some advantage in any particular
enzymatic reaction that proceeds via covalent
catalysis. This reaction is catalyzed by
pyridine, a better nucleophile than water
(pKa5.5). Hydrolysis is accelerated because of
charge loss in the transition state.
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Acetoacetate Decarboxylase
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Acetoacetate Decarboxylase Mechanism
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Lysozyme

• Lysozyme is a small globular protein composed of
  129 amino acids.
• It is also an enzyme which hydrolyzes
  polysaccharide chains, particularly those found
  in the peptidoglycan cell wall of bacteria. In
  particular, it hydrolyzes the glycosidic bond
  between C-1 of N-acetyl muramic acid and C-4 of
  N-acetyl glucosamine.
• It is found in many body fluids, such as tears,
  and is one of the bodys defenses against
  bacteria.
• The best studied lysozymes are from hen egg
  whites and bacteriophage T4.
• Although crystal structures of other proteins had
  been determined previously, lysozyme was the
  first enzyme to have its structure determined.

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Lysozyme Active Site
The X-ray crystal structure of lysozyme has been
determined in the presence of a non-hydrolyzable
substrate analog. This analog binds tightly in
the enzyme active site to form the ES complex,
but ES cannot be efficiently converted to EP. It
would not be possible to determine the X-ray
structure in the presence of the true substrate,
because it would be cleaved during crystal growth
and structure determination. The active site
consists of a crevice or depression that runs
across the surface of the enzyme. Look at the
many hydrogen bonding contacts between the
substrate and enzyme active site that enables the
ES complex to form. There are 6 subsites within
the crevice, each of which is where hydrogen
bonding contacts with the sugars are made. In
site D, the conformation of the sugar is
distorted in order to make the necessary hydrogen
bonding contacts. This distortion raises the
energy of the ground state, bringing the
substrate closer to the transition state for
hydrolysis.
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General Acid-Base Catalysis in Cleavage by
Lysozyme
At what position does water attack the sugar?
When the lysozyme reaction is run in the presence
of H218O, 18O ends up at the C-1 hydroxyl group
at site D. This suggests that water adds at that
carbon in the mechanism. From the X-ray
structure, it is known that the C-1 carbon is
located between two carboxylate residues of the
protein (Glu-35 and Asp-52). Asp-52 exists in
its ionized form, while Glu-35 is protonated.
Glu can act as a general acid to protonate the
leaving group in the transition state. Asp can
function to stabilize the positively charged
intermediate. Glu then acts as a general base to
deprotonate water in the transition state.
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Importance of Strain in Catalysis
Stable Chair conformation
Distorted boat conformation
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The Serine Proteases

• The serine proteases are a class of enzymes that
  degrade proteins in which a serine in the active
  site plays an important role in catalysis.
• The family includes among many others,
  Chymotrypsin and trypsin, which weve talked
  about, and Elastase.
• All three enzymes are similar in structure, and
  they all have three important conserved
  residuesa histidine, an aspartate, and a serine.
• Chymotrypsin cleaves after mainly aromatic amino
  acids, while trypsin cleaves after basic amino
  acids. Elastase is fairly nonspecific, and
  cleaves after small neutral amino acids. Notice
  how their active sites are suited for these
  tasks.

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Chymotrypsin Mechanism (Step 1)
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Chymotrypsin Mechanism (Step 2)
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Chymotrypsin Mechanism (Step 3)
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Chymotrypsin Mechanism (Step 4)
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Chmyotrypsin Mechanism (Step 5)
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Chymotrypsin Mechanism (Step 6)
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Chymotrypsin Mechanism (Step 7)
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Enzyme Assays

• In order to study enzyme reactions, there needs
  to be an efficient method for determining how
  fast products are produced by the enzyme. This
  is the enzymes activity. It is measured in
  µmolmin-1mol-1 of active site (turnover number,
  or µmolmin-1mg-1 of protein (specific
  activity).
• Measuring the activity of proteases is not
  necessarily straightforward using the normal
  substrates. You could for example, run a gel
  that might separate parent peptides from the
  cleaved peptides. Therefore, enzymologists make
  frequent use of substrate analogs that might aid
  in measuring enzyme activity.
• Serine proteases cleave ester substrates better
  than peptide substrates. p-nitrophenylacetate
  has an advantage in that the cleaved product
  p-nitrophenol is brightly colored yellow. The
  enzyme can therefore be assay in real time.

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Burst Kinetics

• Enzyme reactions are run under pseudo-first order
  kinetics. That is, the substrate concentration
  is so much higher than that of enzyme, that the
  rate of the reaction only depends on the enzyme
  concentration and not that of the substrate. The
  enzyme is considered to be saturated under
  these conditions.
• For all practical purposes, enzyme reactions are
  typically linear from T0, until the substrate
  concentration decreases to below saturation
  level.
• For chymotrypsin assayed with p-nitrophenylacetate
  , the researchers observed a burst of
  p-nitrophenylacetate followed by a linear slower
  phase. At the same time, acetate production
  showed a lag followed by a linear phase having
  the same rate as p-nitrophenolate production.

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The Rate-limiting Step
The observation of burst kinetics is suggestive
of a fast step in catalysis that is followed by a
slower step. The lag phase that is associated
with acetate production suggests that the slow
step (the rate-limiting step) is release of
acetate from the enzyme active site. The rapid
production of p-nitrophenolate suggests that the
fast step (burst phase) is cleavage of
p-nitrophenylacetate. The slow linear phase
represents release of acetate from the active
site. As long as its there, enzyme cannot bind
another substrate to catalyze its cleavage.
Frequently, burst kinetics is associated with
formation of a covalent bond between some portion
of the substrate, and an amino acid in the active
site of the protein. It could also be associated
with a simple slow release of products.
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The Acyl Enzyme Intermediate
Diisopropylflurophosphate is an inhibitor of
chymotrypsin. It diffuses into the active,
wherein a nucleophilic amino acid attacks the
phosphate, releasing fluoride anion. This
results in a covalent bond between the
nucleophile and the inhibitor. It inhibits the
reaction because it blocks entry of normal
substrates. The enzyme-inhibitor adduct is very
stable. Upon hydrolysis of the protein (6 N HCl,
110C) and amino acid analysis on the
hydrolysate, a novel amino acid was isolated. It
was the diisopropylphosphoryl derivative of
serine.
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The Oxyanion Hole

• The tetrahedral intermediate in chymotrypsin,
  which consists of the Ser195 adduct before
  departure of the leaving group, is considered to
  be the transition state intermediate in the
  chymotrypsin reaction. It is high energy because
  there is a carbon surrounded by 3 electronegative
  atoms, one of which bears a negative charge.
• How is it that the enzyme stabilizes this
  transition state intermediate? The backbone
  amides of gly193 and ser195 form an oxyanion
  hole. They loosely hydrogen bond to the carbonyl
  oxygen under attack. Upon formation of the
  tetrahedral intermediate, the resulting
  carbon-oxygen single bond is longer, and the
  negatively charged oxygen is better accommodated
  in the oxyanion hole.