Mechanisms of Enzyme Action

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Mechanisms of Enzyme Action BCH 520 Chemical
reactions of catalysis Effects that are
responsible for the rate acceleration of enzymes
1. Chemical effects a. acid-base catalysis
b. covalent catalysis 2. Binding effects
a. transition state stabilization b.
proximity effect Terms use to describe enzyme
mechanisms 1. nucleophile electron rich
(negative charge or unpaired electrons) 2.
electrophile electron poor (positive charge)
3. leaving group group displaced by
nucleophile 4. transition state unstable
high energy state, between being a substrate
and a product 5. carbanion carbon that
gains a pair of electrons 6. carbonium ion
carbon that loses electron pair 7. free
radical pair of electrons that split up 8.
oxidation loss of electrons a. removal of
hydrogen b. addition of oxygen c. removal
of electrons Transition State and Activation
Energy
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1. rate of reaction depends on frequency of
molecules colliding 2. collision must be
productive a. orientation must be correct
b. sufficient energy produced to approach
product formation transition state 3.
activation energy a. energy necessary to
go from ground state of substrate to
transistion state 4. transition state is
not stable, cannot be isolated or measured only
inferred 5. some reactions have a stable
intermediate which can be isolated these
reactions have two transition states 6.
enzyme catalysts lower the activation energy by
stabilizing transition states a. loss of
entropy of reactants b. positions reactants
correctly--proximity 7. transition state
binds to enzyme more tightly--helps rate
acceleration Chemical Catalysis Acid/base
and covalent can increase rate of reaction
10-100X á active sites are usually
hydrophobic--interior of protein á will be
some polar amino acids and water present in
active site á these amino acids participate
in the chemical catalysis catalytic center o
asp, gly, his, cys, tyr, lys, arg, ser
are found in active sites (KNOW TABLE 6.1)
o the pKa of side chain will vary depending on
protein environment (see Table 6.2)
Acid/Base catalyis--substrate is not covalently
linked to protein á common mechanism á
involve movement of a proton á amino acids
that can donate/accept a proton asp, glu, lys
and his á his is the most common, pKa 6-7
á these amino acids can act as the functinal
equivalent of a srong acid or base o proton
acceptor (B)--cleave OH, NH or CH bonds o
(B) can generate an OH- group from water--cleave
CN bonds o BH can protonate an atom making a
bond more labile Covalent catalysis--substrate
covalently linked to protein á covalent linked
substrate forms reactive intermediate á
reversible--must regenerate enzyme á 20
enzymes use this type of mechanism á side
chains of some amino acids contain nucleophilic
(supply an electron pair negatively charged)
groups that may form covalent bonds to
intermediates during catalysis A--X E U
X--E A (the group X has been moved from
substrate A and is covalently bound to enzyme)

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X--E B U B--X E (the group X has been
transferred to substrate B and the enzyme is now
released)
pH effects á ionizable amino acids in active
site will be affected by pH á pH optimum for
enzyme can give clues about which amino acids are
involved in the mechanism Binding Effects
proximity and transition state stabilization
Proximity Effect-rate increase when two
reactants are brought out of a dilute
environment and placed close together á
increase rate up to 10,000X á enzymes bring
reactants together--this is their function
to provide a surface and correct orientation
for reaction á contributes to the loss of
the substrate's freedom of movement (loss
of entropy) á the more rigidly held the
substrate the greater the increase in rate--see
example in Fig.6.8 á increases chances of
reaction --molecules are closer together on
enzyme surface--becomes a unimolecular
(intramolecular) reaction á specific binding
in a particular orientation places the
molecular orbitals of substrate in the
appropriate positions for reaction á also
orients substrate molecule with respect to
protein side chains in the active site needed
for catalysis á even non-chiral molecules
when bound in an active site may be
oriented a certain way by the enzyme (e.g.,
aconitase) Binding must be specific, but
not too strong--if too strong, the energy
barrier would be too high and reaction would not
take place á interactions in active site o
hydrophobic--most active sites are
hydrophobic with a few polar amino acids
(catalytic sites)
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o charge-charge--these are stronger in the
non-polar environment of the active site--asp,
glu, his, lys, arg o hydrogen bonding between
substrate and sidechains or backbone o van
der Waals--very tightly packed in active site
Transition State Stabilization á strain and
distortion by enzyme force substrate into the
transistion state á maximal interaction of
enzyme occurs when substrate is in transition
state o transition state "fits" into active
site better than substrate o interaction of
transition state occurs with enzyme that does not
occur with substrate due to changes in charge
or charge distribution á the transition state
must be stablized for catalysis to occur--lowers
activation energy á some inhibitors work
by their resemblence to the transition
state of the substrate, but the reaction
cannot be catalyzed--very potent inhibitors o
these inhibitors are important in learning
about active sites and transition states of
substrates (Fig 6.11 is a very good example) á
catalytic antibodies work by raising
antibodies to substances that resemble
transition states EXAMPLES OF CATALYSIS
Enzyme--substrate binding Lock and Key
model-proposed in 1894 by Emil Fischer á the
enzyme and substrate have complimentary
structures and fit together, specificity for
substrate á update to specificity for
transition state substrate is distorted by
enzyme to transition state Examples of
Catalysis Triose phosphate isomerase-interconver
sion of dihydroxyacetone phosphate (DHAP) to
glyceraldehyde-3-Phosphate via a cis-enediol
intermediate á glycolytic enzyme á Moves
the aldehyde bond from C1 to C2. These
are isomers, so TPI is an isomerase á
diffusion controled reaction substrate is
converted to product immediately á rate is
limited only by the finding/binding of substrate
á E DHAP U E-DHAP U E-DHAP U E-G3P U E
G3P á each of these reactions as an
activiation energy, in this case all 4
have approximately the same energy which
make it nearly kinetically perfect enzyme
DHAP Glyceraldehyde-3- P Triose phosphate
isomerase dimer (beta barrel)
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á Active sites are near the top of the
barrel--ligand is shown in cpk colors--turn
molecule on side and look down barrel and see
active site á Glu 165 (green) acts as a BASE
or a proton acceptor and pulls a proton from C2
with the assistance of His 95 (blue)
simultaneously acting as a proton donor or
ACID on C1 á A proton is the returned to C1
by Glu 165 and removed from C2 back to His 95
á Phosphate group is held in place with a salt
bridge between lys12 (yellow) á loop from
AA 166-176 (purple) forms a lid that
prevents the enediol from escaping until
isomerization is complete-mechanism of this
unknown--but an example of proteins being
dynamic! á Protecting enediol intermediate in
the active site prevents reaction with solvent
and degradation into methyl glyoxal and
inorganic phosphate, a more favorable
reaction in solution than isomerization á
mutational analysis has shown how important Glu
165 is--even changing to Asp lowers catalysis
by 1000 fold removal of protective loop allows
enediol to escape and react with solvent
lowering efficiency 100,000 fold! á Enzyme in
this case has two functions-accelerating the
reaction and protection of the intermediate
Lysozyme á discovered by Alexander Fleming
(discovered penicillin later) in nasal mucosa as
having antibacterial properties á present in
secretions (tears, mucosa, etc) and egg
white, as an antibacterial agent á
hydrolyzes glycosidic bond of bacterial cell wall
polysaccharide á cell wall polysaccharide
made up of alternating N-acetyl muramic acid and
N-acetylglucosamine á lysozyme has 6 binding
sites for monomer sugar residues (A,B,C,D,E,F)
á A,C,E accomodate glycosamine (cannot
accomodate muramic acid side chain) á B,F
accomodate muramic acid á D can only bind
muramic acid if distorted from chair (more
stable conformation) to half chair--strain and
distortion á note the groove where the
substrate can bind--proximity and strain á
glu35 (green) and asp52 (blue) are opposite each
other á glu-35 (protonated, shown in green)
acts as an acid catalyst and donates proton to
oxygen in glycosidic bond (see Fig.
6.16)--creates a carbonium ion-- acid/base
catalysis á asp-52 (unprotonated, shown in
blue) forms an ion pair with the carbonium
ion--transition state stabilization á water
comes in to replace cleaved product and
donates hydroxide to the carbonium ion and
proton back to glu-35 á lysozyme shows all
the effects of proximity, acid/base catalysis,
distortion and transition state stabilization
The serine proteases-trypsin and chymotrypsin
á digestive enzymes, synthesized in pancreas

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á Hydrolyze peptide bonds, very specific á
trypsin hydrolyzes to the carboxyl side of lys
and arg á chymotrypsin hydrolyzes to the
carboxyl side of large hydrophobic residues, phe
and tyr á elastase hydrolyzes small
hydrophobic residues, gly, ala á have similar
3D structures and similar mechanism á
specificity arises from "pocket" that holds
substrate, for trypsin, a carboxylate (negative
charge) is in the bottom of the pocket to hold
the positively charged arg or lys á for
chymotrypsin there are hydrophobic residues and
the pocket is wider á elastase has
shallower pocket and "guards" to prevent
larger residues from binding Zymogens á
proteins synthesized in an inactive form and
cleaved by proteases to the active form á
chymotrypsin, trypsin, elastase are all zymogens
á cascade effect, small amount of protease
will activate one protease which in turn will
activate others á have inhibitor proteins
that prevent premature cleavage o binding is
very tight--strongest protein/protein
interactions known o inhibitor protein
mimics substrate, but prevents hydrolysis by
excluding water from the active site
Chymotrypsin á needs 3 cleavages to be fully
active á initial cleavage between amino acids
15-16 á the N terminal peptide remains
attached by disulfide bond á residues 14-15
removed á residues 146-149 removed á
three polypeptides held together by disulfide
bonds á cleavage between 15-16 generates
a new amino terminus that forms a salt
bridge with asp 194 á this salt bridge causes
conformational changes to form the active site
á brings asp102, his57 and ser195 into
proximity for the active site Active site of
serine proteases á catalytic triad asp 102,
his57 and ser195 are connected by hydrogen
bonding á his 57 identified using
irreversible inhibitor p-toluenesulfonyl
phenylalanylglycine--resembles the transition
state o inhibitor is covalently linked to
his57 á asp 102 identified from crytal
structures
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á Ser 195 also in active site--unusual for
serines to be reactive, not very
nucleophilic o proximity to his 57 and asp
102 contribute to its reactivity
á his 57(blue) is polarized by the negative
charge of asp 102 (yellow), making it act as a
base (proton acceptor) and extracts a proton
from ser195 substrate shown in green in picture
on right á the serine is now a nucleophile
and attacks the carbonyl of the peptide chain,
forming the tetrahedral transition state (see
fig. 6.27) á N-terminal portion of the
substrate is covalently bound to enzyme as an
acyl- enzyme intermediate (tetrahedral
intermediate) that is stablized by hydrogen
bonding to other amino acids in the oxyanion hole
á C-terminal portion of the substrate
extracts the proton (originally from the
serine) from the histidine to make a new
amino terminal group---exits the active site
á water comes in to replace the exiting
C-terminal fragment and gives up a proton
to his forms another tetrahedral transition
state also stablized in the oxyanion hole by
hydrogen bonding á his donates a proton back
to serine, second peptide chain released with new
carboxy terminus á chymotrypsin also uses
proximity (steps 1 and 4), acid/base
catalysis (2,4), covalent catalysis (steps 2-5)
and stabliziation of transition state (2,4)