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Abstract:_______________________________________________________________________________
___
Chymotrypsin is a serine protease capable of degrading proteins through peptide bond
hydrolysis. The enzyme cleaves peptide bonds C-terminally to large hydrophobic amino acid
residues such as tyrosine, tryptophan and phenylalanine. Previous in vitro assays of
chymotrypsin activity have shown the proteins ability to cleave ester bonds as well as peptide
bonds. The two reactions occur via production of an acyl enzyme intermediate, which is followed
by deacylation to release the cleaved polypeptide and regenerate the active enzyme. Though
the mechanisms of bond cleavage are analogous, peptide and ester substrates follow different
Michaelis-Menten kinetics, a result of unique rate limiting steps for each substrate. In this
experiment the kinetic and mechanistic parameters of ester cleavage by -bovine chymotrypsin
was followed through spectrochemical activity assays. An activity assay of chymotrypsin
catalyzed cleavage of the ester bond of N-acetyl L-tyrosine ethyl ester (ATEE) was carried out to
determine the kinetic parameters Vmax, Km, and kcat. The activity of chymotrypsin to cleave the
ester bond of para-nitrophenylacetate was then analyzed in a burst hydrolysis assay. The burst
hydrolysis assay was used to determine the relationship of enzyme concentration to the rate of
acyl-enzyme intermediate formation and the rate of enzyme deacylation, and to determine
percent activity of chymotrypsin in a stock solution.
Introduction:___________________________________________________________________________
____
Bovine -chymotrypsin is a digestive enzyme synthesized in its zymogen form,
chymotrypsinogen, by acinar cells in the pancreas. The enzyme cleaves N-terminally to large
hydrophobic residues such as phenylalanine and tyrosine. The inactive zymogen is comprised of
a single polypeptide of 245 amino acids. Upon suitable stimulation chymotrypsinogen is secreted
from the pancreas into the intestine where it is proteolytically cleaved by trypsin into its active
form. All forms of chymotrypsin are serine proteases. Serine proteases act through a catalytic
triad of serine, histadine, and aspartate residues. A serine protease mechanism for chymotrypsin
activity is shown in figure 1.
Figure 1: The catalytic mechanism of the chymotrypsin active site. The reaction begins with
a serine nucleophile attacking the N-terminal peptide bond. This tetrahedral intermediate is
stabilized through interactions with the amide backbone of Gly-193 in the oxyanion hole. The
intermediate collapses with release of a new N-terminal polypeptide and formation of an acyl-
Initial
enzymechymotrypsinogen cleavage occurs
intermediate. Hydrolysis at the
releases the active
peptide bond between
enzyme Arg-15
and cleaved and Ile-16;The
polypeptide. with
further autocatalytic
specificity cleavage is
of chymotrypsin after residues
dictated by13,the146, andhydrophobic
large 147 leading S1-specificity
to the various pocket
isoformsC-of
chymotrypsin.
terminal to the Crystal structures
active site. Theof -chymotrypsin
pocket binds largeshowed autocatalytic
aromatic amino cleavage
acids suchbetween
as
residues 13-14 and 148-149 to release two dipeptides alongside the 13 residue N-terminal
polypeptide chain. Cleavage of chymotrypsinogen induces multiple structural changes leading to
enzyme activation. Cleaving the of the N-terminal change allows the new N-terminal Ile-16 to
form a salt bridge with Asp-194 near the active site. This electrostatic interaction shifts the
backbone of residues 190-195, allowing Gly-193 and Ser-195 to assume the correct formation for
the oxyanion hole. The backbone change also shifts Met-192, which twists 180 allowing
substrate access to the S1 specificity pocket (Figure 2).
Initial studies on chymotrypsin showed a deviation from classic Michaelis-Menten kinetics. Rather
than a classic linear increase in absorbance upon p-nitrophenylacetate hydrolysis (as assumed
by the simple two step enzyme kinetic model)(Figure 3), chymotrypsin hydrolysis shows an initial
burst phase, indicated by an exponential increase in absorbance, followed by a linear steady-
state phase. To rationalize this difference, it was proposed that chymotrypsin hydrolyzes bonds
through a district and isolatable intermediate. Since this proposition, a large body of evidence
has proven the existence of a distinct intermediate where residue Ser-198 is acylated with the C-
terminal half of the peptide substrate.
From previous studies on chymotrypsin activity, it has been shown that the enzyme has the
potential to cleave ester as well as peptide bonds. However, these hydrolysis reactions follow
different kinetic pathways due to unique rate limiting steps in the reaction mechanism.
Materials and
Methods______________________________________________________________________
All spectroscopic readings were carried out using a Biowave II spectrophotometer and in 3 ml
silica cuvettes of 1 cm pathlength.
Difference Extinction Coefficient Analysis of ATEE and AT:
Sample solutions of ATEE and AT at 1.0, 0.8, 0.5, 0.4, and 0.3 mM concentrations were prepared
through dilution of a 5mM stock solution in 100mM Tris-HCl buffer (pH: 8.0). The spectrometer
was calibrated with 3.0 ml Tris-HCl buffer and the absorbance values of 1.0 mM ATEE and AT
were recorded from 200-400 nm. From the reading, 235 nm showed the largest absorbance
difference between the two compounds. The absorbances of each sample solution were then
taken at 235 nm to determine a difference extinction coefficient between the two species ().
Each 235 nm absorbance reading was carried out three times.
Results:_________________________________________________________________________
0.20000000
0.15000000
0.10000000
0.05000000
0.00000000
Absorbance Difference (Au)
-0.05000000
-0.10000000
-0.15000000
-0.20000000
-0.25000000
-0.30000000
Wavelength (nm)
Concentration (mM)
1/[ATEE]
Burst Hydrolysis of p-
Figure 4: The Beer-Lambert plot of various p-nitrophenol
Nitrophenylacetate:
solutions at 405 nm. The extinction coefficient was
Figure 3: Lineweaver-Burke plot ofdetermined
the ATEE as the slope of the best fit line through the
hydrolysis
assay. Weighted and unweighted origin. Error bars
least squares are too small to appear on the graph.
analysis
was carried out to determine the kinetic parameters of
chymotrypsin catalyzed ATEE hydrolysis.
Upon the addition of substrate to each chymotrypsin solution a rapid increase in absorbance at
405 nm was observed. This burst
phase was quickly succeeded by a Table 3: The deacylation rate of each burst hydrolysis
linear increase in assay alongside standard deviations and standard errors.
Deacylation Rateconcentrations
Enzyme Avera Standa Standa
shown are after dilution of the 20l
absorbance,
[E] samples with 3 ge
ml rd
solutions of rd
substrate and buffer.
corresponding to
mg/ml Deviati Error
the deacylation
on
rate. By extrapolating
0.044 0.039 0.038 0.040
the linear phase 0.0062 0.0032 0.0018 back to time
3 1 6 7
zero a burst height was
0.083 0.068 0.081 0.077
recorded for 0.0123 0.0078 0.0045 each enzyme
0 8 3 7
solution. The burst heights
and deacylation 0.0185 0.151 0.139 0.136 0.142 rates of each
0.0081 0.0047
enzyme solution 4 6 0 3 are shown in
Table 5 and Table 0.0246 0.177 0.208 0.200 0.195 0.0157 0.0091 6. The
dependence of 7 0 3 3 burst height
and deacylation 0.0308 0.228 0.227 0.242 0.232 0.0085 0.0049 rate on enzyme
concentration is 3 8 7 9 shown in Figure
5 and Figure 6 respectively. From the data in Figures 5 and 6 it is shown that both burst height
and deacylation rate are linearly dependent on enzyme concentration.
Table 4: The burst height of each burst hydrolysis assay
alongside standard deviations and standard errors. Enzyme
concentrations shown are after dilution of the 20 l samples
[E] 3 ml solutions
with Burst Height Avera
of substrate and Standa Standa
buffer.
mg/ ge rd rd
ml Deviati Error
on
0.00 0.10 0.089 0.166 0.120
0.0411 0.0237
62 37 9 9 2
0.01 0.16 0.149 0.196 0.170
0.0238 0.0138
23 55 9 7 7
0.01 0.24 0.188 0.201 0.210
0.0270 0.0156
85 05 9 2 2
0.02 0.22 0.259 0.280 0.255
0.0267 0.0154
46 78 2 8 9
0.03 0.27 0.326 0.316 0.304
0.0293 0.0169
08 12 0 8 7
0.250
0.200
0.150
0.050
0.000
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0.018
0.016 f(x) = 0.57x
0.014
0.012
0.010
BUrst Height (mM) 0.008
0.006
0.004
0.002
0.000
0.000 0.010 0.020 0.030 0.040
Discussion_____________________________________________________________________________
____
Chymotrypsin catalyzed hydrolysis of amide and ester substrates follows two kinetic pathways; a
consequence of the unique chemical properties of amide and ester bonds. The rate-limiting step
of amide hydrolysis is formation of the acyl enzyme intermediate, whereas the rate-limiting step
of ester hydroylsis is enzyme deacylation. Early experiments on the hydrolysis of ester and
amide compounds determined ester substrates more reactive toward weak nucleophiles
compared to amides. This difference arises from the more stable resonance form of the amide
bond, which places a positive charge on nitrogen. The analogous ester resonance form places a
positive charge on oxygen, which is marginally destabilizing. As amide bonds are less susceptible
to hydrolysis it follows that the rate limiting step of chymotrypsin catalyzed hydroylsis is
formation of the acyl-enzyme intermediate, as this step requires breaking of the N-C bond.
Studies of chymotrypsin activity on ester studies showed that the rate did not depend on the
nature of the leaving group produced during formation of the acyl-enzyme intermediate. Less
stable methoxy leaving groups showed little rate change compared to more stable ethoxy
leaving groups. This research provided initial evidence for the rate-limiting step of ester
hydrolysis as deacylation.
Chymotrypsin