Peptidases/Proteases
Proteases catalyze the hydrolysis of peptide bonds, in which a molecule of water attacks the carbonyl
group of the peptide bond to generate carboxylic acid and free amine groups. Proteases can be
exopeptidases, in which the N-terminal or C-terminal amino acid is hydrolyzed from the peptide
chain, or endopeptidases, in which cleavage takes place between two internal amino acids of the
peptide chain. Proteases can be non-specific with respect to the peptide bonds that they hydrolyze, or
they can display substrate selectivity.
O
H2 N
CO2 O H2 C
+H N
3
H2 C
H
H
N
H
H
N
O H2 C
H3 C
CH3
O CH H
H
N
H
CH2
O H2 C
H
N
O H2 C
CH2
CH
H3 C
H
N
H
CH3
H
H
N
O H2 C
O H3 C
H
H
N
H
H
N
O
OH
H2 C
CH2
+H N
3
leucine amino peptidase
OH
carboxypeptidase A
trypsin
chymotrypsin
Chymotrypsin cleaves peptides that contain
phenylalanine and tyrosine residues. It is also decently
active toward peptides containing tryptophan.
Peptidases/Proteases
The potential sites of interaction of the substrate with the enzyme
are designated P (shown in red), and corresponding binding sites on
the enzyme are designated S. The scissile bond (also shown in red)
is the reference point.
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, trypsin, elastase, and
subtilisin.
The first three enzymes have similar
structures, and they all have three important
conserved residues–a histidine, an aspartate,
and a serine. Subtilisin is structurally
dissimilar but has the catalytic triad
identically positioned in its active site.
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.
Chymotrypsin Binding Pocket
It is S1 that is extremely important in substrate specificity. In the case of
elastase, which has a less deep S1 subsite, S2-S5 are also very important in
bringing about binding of the substrate.
P2
S3
H
N
CH
O
S1
R2
N
H
H
N
CH
R3
P3
P1'
O
S2
CH
O
S2'
R1'
N
H
H
N
CH
R1
P1
P3'
O
S1'
CH
O
R3'
N
H
R 2'
P2'
S3'
Leaving Group
The binding pocket for aromatic side chains of the specific substrates of chymotrypsin is a well-defined
slit in the enzyme 10 to 12 angstroms deep and 3.5 to 4 by 5.5 to 6.5 angstroms in cross section. An
aromatic ring is about 6.5 angstroms wide and 3.5 angstroms deep. Chymotrypsin will only cleave
peptides containing L-amino acids. The closer to the scissile bond, the more important the binding
interactions. The enzyme is more tolerant of variations in the structures of the amino acids in the leaving
group than in the acyl group. It will tolerate almost any acyl derivative that is subject to chemical
hydrolysis. Esters and thioesters are cleaved faster than amides, as they are in nonenzymatic, basecatalyzed hydrolysis.
Chymotrypsin Binding Pocket
The hydrophobic pocket of
chymotrypsin is responsible
for its substrate specificity.
The key amino acids that
constitute the binding site are
labeled, including the activesite serine residue (boxed).
The position of an aromatic
ring bound in the pocket is
shown in green.
The Substrate Specificity of Chymotrypsin
Based on S1-P1 Complementarity
Chymotrypsin Mechanism
Chymotrypsin Mechanism (Step 1)
Chymotrypsin Mechanism (Step 2)
Chymotrypsin Mechanism (Step 3)
Chymotrypsin Mechanism (Step 4)
Chmyotrypsin Mechanism (Step 5)
Chymotrypsin Mechanism (Step 6)
Chymotrypsin Mechanism (Step 7)
Chymotrypsin Mechanism
Enzyme Assays/Special Substrates
•
•
•
•
•
What’s the evidence for the mechanism of
chymotrypsin?
In order to study enzyme reactions, one needs an
efficient method for determining how fast products
are produced by the enzyme. This is the enzyme’s
activity. It is reported in mol product/(min•mol
enzyme) (turnover number).
Review of kinetics of enzyme catalysis, meaning of
kcat, KM.
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 or
changing the rate-determining step in order to
uncover intermediates. In 1954, Hartley and Kilbey
used p-nitrophenylacetate as a substrate for
chymotrypsin, as an outgrowth of earlier experiments
concerning the inhibition of esterases by
organophosphorous compounds.
Serine proteases cleave ester substrates better than
peptide substrates. p-nitrophenylacetate has an
advantage in that the cleaved product pnitrophenolate is brightly colored yellow. the
extinction coefficient for p-nitrophenylacetate is
e412=14,000 M-1 cm-1.
Enzyme Assays/Special Substrates
Burst Kinetics
•
•
Enzyme reactions are run with substrate
in vast excess of enzyme. With
sufficiently high [S], the enzyme is
“saturated” and activity becomes
independent of [S], i.e. linear with time or
zero order until the substrate
concentration decreases to below
saturation level.
In assay of chymotrypsin with pnitrophenylacetate, a very poor substrate,
Hartley and Kilbey had to use high
enzyme concentration. They observed a
burst of p-nitrophenylacetate followed by
a linear slower phase. Later, acetate
production was shown to exhibit a lag
followed by a linear phase having the
same rate as p-nitrophenolate production.
Amplitude of the Burst
•
•
•
Hartley and Kilby found that when they
extrapolated the slow phase to t = 0, the
magnitude of the burst was equal to the
enzyme concentration.
Assays containing greater amounts of
enzyme led to larger bursts, while those
containing lesser amounts of enzyme
led to smaller bursts. This showed that
the burst was not an artifact, as it was
dependent on the enzyme
concentration.
From these data, Hartley and Kilby
suggested that the cleavage of pnitrophenolacetate by chymotrypsin
occurs in two steps. The first step is a
rapid acylation of some group on the
enzyme, while the second is hydrolysis
of the acyl enzyme.
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 (not carried out in original
experiment) 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 it’s 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 (covalent catalysis). It
could also be associated with a simple slow release of bound products.
Catalysis of the Rate-limiting Step
Hydrolysis of N-acetylphenylalanyl-chymotrypsin intermediate is ~
105-fold faster than hydrolysis of acetyl-chymotrypsin intermediate.
Why?
Criteria for Covalent Catalysis
•
There are several criteria that must be satisfied in order to prove a
mechanism involving covalent catalysis.
•
1. The covalent enzyme-substrate complex must be isolated,
chemically characterized, and shown to be present during turnover.
2. Chemical and kinetic competence must be established. It must be
shown that the intermediate, when re-introduced into the reaction, will
react to give normal products at a rate that is at least as fast as kcat.
•
•
3. When covalent enzyme-substrate intermediates are first observed
with poor substrates, it is important to demonstrate that the normal
substrates react by the same mechanism.
The Acyl Enzyme Intermediate
Diisopropylfluorophosphate is an inhibitor of chymotrypsin. It diffuses into the active site, 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, 110°C) and
amino acid analysis of the hydrolysate, a novel amino acid was isolated. It was shown to be
phosphoserine.
Evidence for Covalent Adduct
CH3 O
H3C C
14
C Enzyme
CH3
3
H
CH3 O
H3C C
14
C O
NO2
+
+ Enzyme
CH3
3
H
p-nitrophenyltrimethylacetate leads to formation of an
exceptionally stable adduct. This is probably due to steric
hindrance as well as an inductive effect of the methyl
groups. This intermediate has been isolated and
crystallized on the enzyme. It hydrolyzes slowly, and on
storage, the enzyme becomes active with time.
Deacylation can be enhanced with stronger nucleophiles,
such as hydroxylamine (1 M), which forms the acyl
hydroxamic acid.
O
NO2
Evidence for Covalent Adduct
CH3 O
H3C C
14
C Enzyme
CH3
3
H
CH3 O
H3C C
14
C O
NO2
+
+ Enzyme
CH3
3
H
p-nitrophenyltrimethylacetate leads to formation of an
exceptionally stable adduct. This is probably due to steric
hindrance as well as an inductive effect of the methyl
groups. This intermediate has been isolated and
crystallized on the enzyme. It hydrolyzes slowly, and on
storage, the enzyme becomes active with time.
Deacylation can be enhanced with stronger nucleophiles,
such as hydroxylamine (1 M), which forms the acyl
hydroxamic acid.
O
NO2
Evidence for Covalent Adduct: Isolation by
Gel-Filtration Chromatography
•
•
•
A dual label technique can be used, in which 3H and
14C can be quantified simultaneously by scintillation
counting. This allows both portions of the substrate to
be followed during catalysis. By knowing the original
specific activity of the substrate (dpm/µmol) the
substrate can be quantified.
Enzyme-bound radioactivity can be separated from
small molecules (substrate) by gel-filtration, in which
molecules migrate through a matrix based on size. For
example, G-25 will exclude most globular molecules
having a molecular weight greater than 5000. They
will flow directly through the column without
retardation. Small molecules, however, will be
retarded because they enter the beads of the matrix.
Chymotrypsin was treated with p-nitrophenylacetate at
pH 5 (pH optimum = 8) to slow down all of the steps in
the reaction. Inactive and radiolabeled chymotrypsin
was produced. Upon raising the pH to 8, all of the
radioactivity was released at a rate that was equal to the
rate of hydrolysis at pH 8. This suggests kinetic
competence.
Evidence for Covalent Adduct
CH3 O
H3C C
14
C Enzyme
CH3
3
H
CH3 O
H3C C
14
C O
NO2
+
+ Enzyme
CH3
3
H
p-nitrophenyltrimethylacetate leads to formation of an
exceptionally stable adduct. This is probably due to steric
hindrance as well as an inductive effect of the methyl
groups. This intermediate has been isolated and
crystallized on the enzyme. It hydrolyzes slowly, and on
storage, the enzyme becomes active with time.
Deacylation can be enhanced with stronger nucleophiles,
such as hydroxylamine (1 M), which forms the acyl
hydroxamic acid.
O
NO2
Indirect Criteria for Covalent Catalysis
•
H
O
C
C
HN
N
H
X
–OEt!
–OMe!
–OPNP!
–NH2!
!!
!Km (M)!
9.7 x 10-5!
9.5 x 10-5!
0.2 x 10-5!
500 x 10 -5!
C
X
O
•
H3C
•
Turnover number (min-1 ) •
26.9
27.7
30.5
0.036
The rate-determining step for a
substrate that is not particularly good
(p-nitrophenyl acetate), was shown to
be hydrolysis of the acyl enzyme
intermediate. Here, a molecule that
better mimics the actual substrate was
used to study catalysis by
chymotrypsin.
Notice that all of the ester substrates of
N-acetyl-tryptophan are cleaved with
similar turnover numbers. Notice also
their Km values.
Notice that kcat for the amide is
significantly less than that of the esters,
and that the Km value for the substrate
is significantly higher.
We know from our burst kinetics
experiments that with ester substrates,
the rate determining step is hydrolysis
of the acyl enzyme intermediate. This
is an intermediate that should be
common to all substrates (amides or
esters), and should be cleaved with
similar rate constants. This suggests
that amide hydrolysis proceeds by a
different mechanism, or that there is a
change in the rate-determining step.
Change in Rate Limiting Step
E+S
k1
k-1
k2
ES
k3
ES'
E+P
P1
v=
k2k3
[Et][S]
What is KM?
k2 + k3
k-1 + k2
k1
What is kcat?
k3
k2 + k3
+
[S]
Indirect Criteria for Covalent Catalysis
•
H
O
C
C
HN
N
H
X
–OEt!
–OMe!
–OPNP!
–NH2!
!!
!Km (M)!
9.7 x 10-5!
9.5 x 10-5!
0.2 x 10-5!
500 x 10 -5!
C
X
O
•
H3C
•
Turnover number (min-1 ) •
26.9
27.7
30.5
0.036
The rate-determining step for a
substrate that is not particularly good
(p-nitrophenyl acetate), was shown to
be hydrolysis of the acyl enzyme
intermediate. Here, a molecule that
better mimics the actual substrate was
used to study catalysis by
chymotrypsin.
Notice that all of the ester substrates of
N-acetyl-tryptophan are cleaved with
similar turnover numbers. Notice also
their Km values.
Notice that kcat for the amide is
significantly less than that of the esters,
and that the Km value for the substrate
is significantly higher.
We know from our burst kinetics
experiments that with ester substrates,
the rate determining step is hydrolysis
of the acyl enzyme intermediate. This
is an intermediate that should be
common to all substrates (amides or
esters), and should be cleaved with
similar rate constants. This suggests
that amide hydrolysis proceeds by a
different mechanism, or that there is a
change in the rate-determining step.
Change in Rate Limiting Step
E+S
k1
k-1
k2
ES
k3
ES'
E+P
P1
v=
k2k3
[Et][S]
What is KM?
k2 + k3
k-1 + k2
k1
What is kcat?
k3
k2 + k3
+
[S]
Trapping of an Intermediate
"Acyl Enzyme"
O
R
H2N
C
X
O
NH2
C
C
Enzyme
HX Enzyme
O
CH 3
H
H2N
L-alanine amide
R
C
O
NH
C
C
CH 3
H
Better acceptor. Amines are good nucleophiles, and acceptor binds to
the enzyme in the site that is occupied by the leaving groupd during
the hydrolysis of a peptide.
O
k3
Acyl enzyme
R
H2 O
C
OH
+
k4
R'NH2
HX
-d[ES] = (k3 [H2 O] + k4 [R'NH2])[ES]
dt
O
H2N
R
C
O
NH
C
C
H
Enz
CH 3
+
HX
Enz
Partitioning of a Common Intermediate
O
H 3C
C
O
N
H
CH C
OH
CH 2
O
H 3C
C
O
N
H
CH C
OCH 3
69%
CH 2
O
H 3C
C
H2O
O
N
H
CH C
NM e3
N
H
CH 2
67%
O
H 3C
C
O
N
H
CH C
X
Enz
CH 2
O
H 3C
C
O
N
H
CH C
N
H
NM e2
CH 2
63%
O
H 3C
C
O
N
H
CH C
CH 2
O
N
H
H
C
C
H 2N
NH 2
O
NH 2
C
C
CH 3
H
CH 3
67%
O
H 3C
C
O
N
H
CH C
CH 2
H
N
O
NH 2
C
C
H
CH 3
Probing The Catalytic Triad by pH-Rate Profiles
(Frey and Hegeman, pp. 112-116)
pH-Rate Profiles of Enzymes (Frey and Hegeman, pp. 112-116)
pH-Rate Profiles of Enzymes (Frey and Hegeman, pp. 112-116)
pH-Rate Profiles of Enzymes (Frey and Hegeman, pp. 112-116)
Probing The Catalytic Triad by pH-Rate Profiles
(Frey and Hegeman, pp. 112-116)
Caution Against Over-interpretation of pH-Rate Profiles (Frey and
Hegeman, pp. 112-116)
The Oxyanion Hole
•
•
•
•
The tetrahedral adduct between Ser195 and
the scissile peptide is considered to be an
intermediate in the chymotrypsin reaction. It
has relatively high energy because there is a
carbon surrounded by 3 electronegative
atoms, one of which bears a negative charge.
The high-energy intermediate should closely
resemble the transition state for the initial
attack.
Stabilization of the intermediate/transition
state should accelerate the attack.
How is it that the enzyme stabilizes this
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 carbonoxygen single bond is longer, and the
negatively charged oxygen is better
accommodated in the oxyanion hole.
The Oxyanion Hole
•
•
•
•
The tetrahedral adduct between Ser195 and
the scissile peptide is considered to be an
intermediate in the chymotrypsin reaction. It
has relatively high energy because there is a
carbon surrounded by 3 electronegative
atoms, one of which bears a negative charge.
The high-energy intermediate should closely
resemble the transition state for the initial
attack.
Stabilization of the intermediate/transition
state should accelerate the attack.
How is it that the enzyme stabilizes this
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 carbonoxygen single bond is longer, and the
negatively charged oxygen is better
accommodated in the oxyanion hole.
Divergent Evolution of Serine Proteases with Related
Tertiary Structures
An overlay of the structure of
chymotrypsin (red) on that of trypsin
(blue) shows the high degree of
similarity. Only α-carbon atom positions
are shown. The mean deviation in
position between corresponding αcarbon atoms is 1.7 Å.
Convergent Evolution of the Asp-His-Ser Catalytic
Triad in Proteases with Unrelated Tertiary Structures
Subtilisin
Subtilisin
The Functionally Conserved Asp-His-Ser Catalytic
Triad in the Structurally Dissimilar Subtilisin
The peptide bond attacked by nucleophilic serine 221 of the catalytic
triad will develop a negative charge, which is stabilized by enzyme
NH groups (both in the backbone and in the side chain of Asn 155)
located in the oxyanion hole.
Dissection of the Contributions to Catalysis in
Subtilisin by Site-Directed Mutagenesis
Residues of the catalytic triad were mutated to alanine, and the activity of the mutated enzyme
was measured. Mutations in any component of the catalytic triad cause a dramatic loss of
enzyme activity. Note that the activity is displayed on a logarithmic scale. The mutations are
identified as follows: the first letter is the one-letter abbreviation for the amino acid being
altered; the number identifies the position of the residue in the primary structure; and the second
letter is the one-letter abbreviation for the amino acid replacing the original one.