Two Substrate Reactions
• Many enzyme reactions involve two or more
substrates. Though the Michaelis-Menten equation
was derived from a single substrate to product
reaction, it still can be used successfully for more
complex reactions (by using kcat).
Random
Ordered
Ping-pong
Two Substrate Reactions
• In random order reactions, the two substrates
do not bind to the enzyme in any given order; it
does not matter which binds first or second.
• In ordered reactions, the substrates bind in a
defined sequence, S1 first and S2 second.
• These two reactions share a common feature
termed a ternary complex, formed between E,
ES1, ES2 and ES1S2. In this situation, no product
is formed before both substrates bind to form
ES1S2.
Two Substrate Reactions (cont)
• Another possibility is that no ternary
complex is formed and the first substrate
S1 is converted to product P1 before S2
binds. These types of reactions are
termed ping-pong or double
displacement reactions.
The catalytic mechanism of
chymotrypsin: a member of the serine
protease family; catalyzes the
hydrolytic cleavage of peptide bonds
adjacent to aromatic amino acid
residues (with a rate enhancement of
at least 109).
Principles illustrated:
Transition-state stabilization;
General acid-base catalysis;
Covalent catalysis.
Chymotrypsin (and other proteins)
are activated via proteolytic cleavage
of precursor proteins (zymogens or
preproteins).
Many proteases activated this way
can be inactivated by inhibitor
proteins tightly-bound in the active
sites.
Active chymotrypsin and
trypsin are produced from
inactive zymogens via
proteolytic cleavage, with
conformational changes
exposing the active sites.
The catalytically important groups of
chymotrypsin were identified by
chemical labeling studies
• Organic fluorophosphates such as
diisopropylphosphofluoridate (DIPF)
irreversibly inactivate chymotrypsin (and other
serine proteases) and reacts only with Ser195
(out of the 25 Ser residues).
A second catalytically important residue, His57,
was discovered by affinity labeling with tosylL-phenylalanine chloromethylketone (TPCK)
• TPCK alkylates His 57
• Inactivation can be inhibited by bphenylpropionate (competitive
inhibitor)
• TPCK modification does not occur
when chymotrypsin is denatured in
urea.
Rapid initial burst kinetics indicates an
acyl-enzyme intermediate
• The kinetics of chymotrypsin is worked out
by using artificial substrates (esters),
yielding spectroscopic signals upon
cleavage to allow monitoring the rate of
Colorless substrate
Yellow product
reactions.
Fast
This reaction is far slower
than the hydrolysis of peptides!
Slow
Km = 20 mM
Kcat = 77 s-1
The catalysis of chymotrypsin
is biphasic as revealed
by pre-steady state kinetics
Slow phase (enzymes will be
able to act again only after a slow
deacylation step)
“burst” (fast) phase (rapid acylation of all
Enzymes leading to release of p-nitrophenol)
Milliseconds after mixing
Determination of the
crystal structure of
chymotrypsin (1967)
revealed a catalytic triad:
195
57
102
Ser , His , Asp .
Chymotrypsin: three polypeptide
chains linked by multiple disulfide
bonds; a catalytic triad.
Active site
His57
Asp102
Cleft for binding
extended substrates
Trypsin, sharing a 40% identity with
chymotrypsin, has a very similar structure.
Ser195
A catalytic triad has been found in all serine
proteases: the Ser is thus converted into a potent
nucleophile
The Peptide Bond has partial (40%) double bond
character as a result of resonance of electrons
between the O and N
The hydrolysis of
a peptide bond
at neutral pH
without catalysis
will take ~10-1000
years!
Chymotrypsin (and other serine
proteases) acts via a mixture of
covalent and general acid-base
catalysis to cleave (not a direct
attack of water on the peptide
bond!)
Asp102 functions only to orient His57.
E
Formation of the
ES complex
S
Formation of ES1
The peptide bond to be
cleaved is positioned by the
binding of the side chain of an
adjacent hydrophobic residue
in a special hydrophobic
pocket.
ES1
ES1
Pre-acylation
oxyanion hole
His57 acts as a general base in
deprotonating Ser195, the alkoxide
ion then acts as a nucleophile,
attacking the carbonyl carbon.
Ser195 forms a covalent bond
with the peptide (acylation) to
be cleaved. a trigonal C is
turned into a tetrahedral C.
The tetrahedral oxyanion
intermediate is stabilized by
the NHs of Gly193 and Ser195
Preferential binding of the transition state:
oxyanion hole stabilization of the negatively
charged tetrahedral intermediate of the
transition state.
His57 acts as a general acid
in cleaving the peptide bond.
ES1
Acylation
Releasing of P1
Acyl-E
The amine product is
then released from the
active site with the
formation of an acyl-enzyme
covalent intermediate.
Entering of
S2
E’S2
Water (the second substrate)
then enters the active site.
Acyl-E
His57 acts as a general base
again, allowing water to attack
the acyl-enzyme intermediate,
forming another tetrahedral
oxyanion intermediate, again
stabilized by the NHs of Gly193
and Ser195 (similar to step 2)
Pre-deacylation
E’S2
EP2
Deacylation
His57 acts as a general acid
again in breaking the covalent
bond between the enzyme
and substrate (deacylation)
(similar to Step 3).
Recovered enzyme
Release of P2
E
EP2
The second product
(an acid) is released
from the active site,
with the enzyme recovered
to its original state.
2nd product
1st substrate
E
EP2
The proposed complete
catalytic cycle of
chymotrypsin
(rate enhancement: 109)
A Ping-Pong Mechanism
ES
1st product
E’S2
Deacylation
phase
Acyl-E
2nd substrate
Acylation
phase