Download A. Reaction Mechanisms and Catalysis (1) proximity effect (2) acid

(P&S Ch 5; Fer Ch 2, 9; Palm Ch 10,11; Zub Ch 9)
A. Reaction Mechanisms and Catalysis
(1) proximity effect
(2) acid-base catalysts
(3) electrostatic
(4) functional groups
(5) structural flexibility
B. Active Site Investigations
(1) kinetic studies
(2) detection of intermediates
(3) x-ray crystallographic studies
(4) chemical modification of amino acid side chains
(5) site-directed mutagenesis studies
C. Specific enzymes
(1) lactate dehydrogenase and alcohol dehydrogenase
(2) ribonuclease A
(3) triose phosphate isomerase
(4) amino acyl tRNA synthetases
(5) carbonic anhydrase
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(1) Proximity effect--enzymes bring reacting species close together
-an enzyme can accelerate a rxn between two species simply by
holding the two reactants close together in an appropriate
orientation
-intramolecular rxns between groups that are tied together in a
single molecule are faster than the corresponding intermolecular
rxns between two independent molecule, eg., cyclization of
succinic acid to form succinyl anhydride c.f. formation of acetic
anhydride from two molecules of acetic acid
-difference in rates are 3-4 orders of magnitude
(intramolecular> intermolecular)
-effect is due mostly to the differences between the entropy
changes that accompany the inter- and intramolecular rxns
-formation of P involves a much larger loss of translational and
rotational entropy in the intermolecular rxn
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-a negative change in entropy increases both the overall free
energy change in the rxn (ΔG = ΔH -TΔS) and the activation free
energy (ΔG‡ = ΔH‡ - TΔS‡) for the formation of the transition
state
-in the intramolecular rxn much of this entropy decrease has
already occurred during the preparation of the reagent
-enzymes that take catalyze intermolecular rxns take advantage of
the proximity effect by binding the reactants close together in the
active site, so that the reactive groups are oriented appropriately
for the rxn
-once the substrates are fixed this way the rxn behaves kinetically
like an intramolecular process
-the entropy decrease associated with the formation of the
transition state has been moved to an earlier step, the binding of
the substrates to form the E-S complex
(2) Acid-Base Catalysis
-task of a catalyst is to make a potentially reactive group more
reactive by increasing its intrinsic electrophilic or nucleophilic
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character
-simplest way is to add or remove a proton eg., hydrolysis of an
ester
-hydrolysis of an ester in neutral aqueous solution can occur if the
O atom of H2O, acting as a nucleophile, attacks the positively
charged carbon
-initial product is an intermediate where the carbon atom has four
substituents in a tetrahedral arrangement
-rxn is completed by the rapid breakdown of the tetrahedral
intermediate to release the alcohol
-H2O is intrinsically a comparatively weak nucleophile, and its
rxn with esters in the absence of a catalyst is very slow
-hydrolysis of esters occurs much more rapidly at high pH, when
the negatively charged OH- replaces H2O as the reactive
nucleophile
-the nucleophilic character of H2O itself can be increased by
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interaction with a basic group other than OH- where the base
offers a pair of electrons to one of the protons of the H2O and
increases the electron density of the oxygen
-general base--used to describe any substance that is capable of
binding a proton in aqueous soln
-enzymes use a number of functional groups to fill this role
(necessary since the [OH-] at physiological pH limits its
availability]
-only requirement is that the base start out in the unprotonated
form, which means that the ambient pH must be above the pKa
of the conjugate acid
-candidates include: basic groups from the ionizable or polar
amino acid side chains, an amino-terminal NH2 group, a
carboxyl-terminal carboxylate ion, or from the oxygen or
nitrogen atom of a peptide bond
-the pKa of these groups can vary over a considerable range →
environment
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-using a general base rather than OH- has the advantage that the
basic group that is provided by the protein can be positioned
precisely with respect to the substrate in the active site,
allowing the proximity effect to come into play
-hydrolysis of an ester can also be catalyzed by an acid where
the acid donates a proton to the oxygen of the ester's carbonyl
group, increasing the positive charge on the carbon and
increasing the susceptibility of the ester to attack by a
nucleophile
-general acid--any substance that is capable of releasing a
proton, and enzymes almost always use such proton donors in
preference to H+ or OH- because a general acid can operate at
moderate pH and is easy to fix in position
-requirement is that the pH be below the pKa
(3) Electrostatic interactions --enzymes act by stabilizing the
distribution of electrical charge in transition states
-suppose the active site of an enzyme included a positively
charged amino acid side chain, such as Lys or Arg, located near
the oxygen atom of a carbonyl group. A fixed positive charge
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in this region would favor the formation of the tetrahedral
intermediate, even if there were no transfer of a proton from the
charged species to the oxygen. A fixed negative charge in the
region of the nucleophile would have a similar effect. The
interactions of such fixed charges are termed electrostatic
effects.
-as a reacting substrate is transformed into a transition state, the
changing charges on its atoms interact with the charges on all of
the other atoms in the surrounding protein, and also with the
charges on any nearby water molecules
-the energy difference between the initial state and the transition
state thus depends critically on the details of the protein
structure
-modern computational techniques, when taken with the wealth of
structural information from X-ray crystallography and other
biophysical studies, have made it possible to calculate the
contributions that various components of an enzyme's active
site make to the activation free energy (ΔG‡), and to predict
quantitatively how ΔG‡ might be altered by modifications of the
protein
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-can be tested experimentally by modifying the gene that
encodes the protein (site-directed mutagenesis)
(4)
Nucleophilic/Electrophilic Catalysis
(a) Nucleophilic Catalysis--strategy is to use stronger
nucleophilic groups (than water) as part of the enzyme's active
site
-HOCH2 group of a serine residue often used as a nucleophile
-instead of immediately yielding the free COOH, the
breakdown
of
the
tetrahedral
intermediate
yields
an
intermediate ester that is covalently attached to the enzyme
-the acyl-enzyme intermediate must be hydrolyzed by a second
rxn in which H2O is the nucelophile
-proteolytic enzymes (serine proteases) work in this
way
-two step pathway requires that the intermediate be more
susceptible to nucleophilic attack by H2O than by the original
ester or amide
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-nucleophilic groups on enzymes participate in a variety of
other types of rxn in addition to hydrolytic rxns, eg., acetoacetic
acid decarboxylase
-rxn proceeds by the formation of a Schiff base intermediate, in
which the substrate is covalently attached to the ε-amino group
of a lysine residue at the enzyme's active site
-this intermediate is formed by a nucleophilic attack of the
amino group on the carbonyl carbon, followed by the splitting
out of H2O
-protonation of the nitrogen atom of the Schiff base introduces a
positive charge that pulls electrons from the nearby carboncarbon bond, causing decarboxylation
-this is an extreme example of an electrostatic effect where the
enzyme introduces a charged group into the substrate itself
-basic feature of this mechanism is the formation of an
intermediate state in which the substrate is covalently attached
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to a nucleophilic group on the enzyme
-the -CH2SH of cysteine is often used as a nucleophile also
-the COOH of Asp and Glu participate in rxns involving the
hydrolysis of ATP and -the imidazole ring of His can play a similar
role
-some enzymes use coenzymes: thiamine, biotin, pyridoxamine, or
tetrahydrofolate as additional nucleophilic reagents
(b) Electrophilic Catalysis--numerous enzymes use bound metal
ions to form complexes with substrates
-metal ion functions as an electrophilic group
-eg., carbonic anhydrase--contains a Zn2+ ion in the active site and
forms a complex with the carbonyl oxygen atom of the aldehyde
or peptide substrate
-withdrawal of electrons by the Zn2+ increases the partial
positive charge on the carbonyl carbon atom and promotes the
rxn of carbon with a nucleophile
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(5) Structural Flexibility--some enzymes undergo major structural
rearrangements when they bind substrates or inhibitors
-eg., hexokinase
ATP + glucose ⇔ ADP + glucose-6-phosphate
-hexokinase
binds
glucose,
it
undergoes
a
structural
organization that brings together the elements of the active site
-the enzyme literally closes like a set of jaws around the
substrate referred to as an induced fit
-carboxypeptidase A undergoes a major structural change when
it binds its substrate
-rearrangement of the protein pulls the hydrophobic part of the
substrate out of the aqueous soln by surrounding it with
nonpolar portions of the protein
-advantages: (1) maximizes the favorable entropy change
associated with removing a hydrophobic molecule from H2O
(2) allows enzyme to intensify the electrostatic effects that
promote the formation of the transition state (the substrate is
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forced to respond to the directed electrostatic fields from the
enzyme's functional groups, instead of the disordered fields
from the solvent
-structural change can help to explain the high specificity of
some enzymatic rxns; eg., hexokinase undergoes a structural
change upon binding glucose which promotes the binding of
the other substrate (ATP)
-ATP doesn't bind unless glucose is already present in the
catalytic site
-if ATP were to bind in the absence of glucose, the enzyme
might have a tendency to catalyze the transfer of phosphate
from ATP to water, resulting in a wasteful loss of ATP:
ATP + H2O → ADP + Pi
-X-ray crystal structure of an enzyme give only static snapshots
of the molecules, which can be very flexible
-vibrations and rotations involving only a few atoms ≡ 10-13 to
10-11 s
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-larger motions, i.e., flipping of aromatic rings ≡ 10-9 to 10-8 s
-major reorganizations ≡ 10-6 to 10-3 s
-all these can be important for catalysis
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