Kinetic Analysis of Bovine -Chymotrypsin Activity on Ester

Substrates
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.

Assay of Chymotrypsin Esterase Activity:

The ATEE solutions prepared above were used in an assay to determine the kinetic parameters
Km, Vmax, and kcat. A 2 mg/ml solution of bovine -chymotrypsin was prepared by dissolving 10
mg protein in 5 ml HCl (10mM). The spectrometer was calibrated with 3.0 ml Tris-HCl buffer and
a 3.0 ml sample of each ATEE solution was then placed in the spectrometer. A 20 l sample of
chymotrypsin solution was added to each sample, quickly stirred, and the absorbance at 235 nm
was taken for 60 s. The spectroscopic readings were carried out three times for each ATEE
solution.

Extinction Coefficient of p-Nitrophenol:

Sample solutions of p-nitrophenol were prepared at 1, 0.8, 0.6, and 0.4 mM concentrations
through dilution of a 1mM stock solution in 50 mM Tris-HCl (pH: 7.8). The spectrometer was
calibrated with 3.0 ml Tris-HCl buffer, and the absorbance of each pNp solution was taken at 405
nm to determine the extinction coefficient at that wavelength. The procedure was carried out
three times for each pNp solution.

Burst Hydrolysis of p-nitrophenol:

A stock 10 mg/ml chymotrypsin solution was used to prepare sample solutions at 10, 8, 6, 4, and
2 mg/ml in 50mM Tris-HCl buffer. A stock solution of the substrate p-nitrophenylacetate was then
prepared by diluting 5 ml of 0.6 mM substrate in 25 ml of Tris-HCl Buffer (pH: 7.8). The
spectrometer was calibrated with 3.0 ml of the pNpa Tris-HCl solution. A 20 l sample of each
chymotrypsin solution was then placed in the spectrophotometer and a 3 ml sample of the pNpa
solution was added. Upon addition of the substrate the absorbance reading at 405 nm was
recorded for 60 s.

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)

Difference Extinction Coefficient Analysis of

ATEE and AT:
To determine a suitable wavelength to follow ATEE hydrolysis by chymotrypsin, the absorbance
difference between ATEE and AT was recorded from 200-400 nm. The absorbance difference
between ATEE and AT is shown in Figure 1. The largest difference (-0.280 absorbance units) is
found at 235 nm, indicating the chymotrypsin catalyzed hydrolysis of ATEE could be followed by
observing the decrease in absorbance at this wavelength.

In order to utilize the absorbance difference at 235 nm a difference extinction coefficient ()

between ATEE and AT was required. The absorbance differences of each ATEE and AT solution
are shown in figure 2. From the data in figure 2, the extinction coefficient of ATEE and AT was
determined by taking the slope linear regression through the origin. This analysis determined
values of 1.780.84 mM-1cm-1 and 1.550.99 mM-1cm-1
respectively. The difference between these two values Figure 1: The absorbance difference of
alongside mathematical error propagation determined the chymotrypsin hydrolysis product AT
the difference extinction coefficient () as 0.24 0.13 and corresponding substrate ATEE. The
mM-1cm-1. graph shows the largest absorbance
difference at 235 nm implicating this as
Assay of Chymotrypsin Esterase Activity: the best wavelength to follow
chymotrypsin esterase activity
spectroscopically.
1.80
1.60 f(x) = 1.79x
1.40 f(x) = 1.55x
1.20
1.00
0.80
Absorbance (235 nm)
0.60
AT Linear (AT) ATEE Linear (ATEE)
0.40
0.20
0.00
0.1 0.3 0.5 0.7 0.9 1.1
0.0 0.2 0.4 0.6 0.8 1.0

Concentration (mM)

Each sample solution of ATEE showed a time-dependent decrease in absorbance at 235 nm

following the addition of chymotrypsin. This absorbance change is consistent with the production
of AT catalyzed by chymotrypsin. The initial gradients of each ATEE assay were recorded
alongside calculated standard deviations and errors (Table 1). From these values a Lineweaver-
Burke plot was created to determine kinetic parameters of ATEE hydrolysis (Figure 3). Using
unweighted and weighted least squares analysis the parameter Vmax was determined as the
inverse of the intercept. Initial analysis gave Vmax in units of Absorbance/min. Dividing Vmax by
the difference extinction coefficient gave Vmax in units of mM/min which was subsequently
converted to mM/s (Table 2). Following the calculation of Vmax, Km was determined as the
quotient of the slope and intercept, and kcat was calculated as the quotient of Vmax and total
enzyme concentration (0.0053 mM).

Table 1: The initial velocities of

Initial Velocity (Au) Averag Standa Standa
chymotrypsin catalyzed ATEE
[ATEE] e rd rd
hydrolysis at different ATEE
mM Velocit Deviati Error
concentrations. Standard errors and
y on
standard deviations of the initial
1.0 0.643 0.659 Figure
0.592 2: The
0.631 absorbance
0.035 difference at 235 nm of
0.020
velocities are also shown
0.8 0.479 0.483 various
0.445 ATEE
0.469 and
0.021 AT concentrations.
0.012 From this
0.5 0.509 0.383 information
0.391 a difference
0.427 0.071 extinction
0.041 coefficient of ATEE
and AT was determined. Error bars are too small to
0.4 0.330 0.366 0.378 0.358
appear on the graph.
0.025 0.015
0.3 0.294 0.294 0.270 0.286 0.014 0.008
 Table 2: 4.00
The kinetic parameters of
ATEE hydrolysis by bovine -
Weighted 3.50
chymotrypsin. All three parameters
Value Error () were determinedf(x) =
f(x) = 0.75x
0.77x +
+ 0.97
0.96
3.00
using weighted and
Vmax (Au/min) 1.036 0.220 unweighted least squares analysis of
Vmax (mM/s) 0.073 0.043 the Lineweaver-Burke
2.50 Unweighted plot shown in Linear (Unweighted)
Km (mM) 0.794 0.211
-1 2.00
kcat (s ) 138.305 81.900 1/Vo
Unweighted 1.50
Value Error ()
1.00
Vmax (Au/min) 1.034 0.213
Vmax (mM/s) 0.073 0.043 0.50 Weighted Linear (Weighted)
Km (mM) 0.771 0.184
-1
0.00
kcat (s ) 138.016 81.397
0.50 1.00 1.50 2.00 2.50 3.00 3.50

1/[ATEE]

Extinction Coefficient of p-Nitrophenol

As the substrate p-nitrophenylacetate shows no absorbance at 405 nm the extinction coefficient

of p-nitrophenol is analogous to the difference extinction coefficient of the above experiment.
The 405 nm absorbance of varying concentrations of p-nitrophenol is shown in Figure 4. Using
the Beer-Lambert equation and taking the slope of the line in Figure 4 the extinction coefficient of
p-nitrophenol at 405 nm was determined to be 19320 1.65 M-1cm-1.

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

Deacylation Rate (Au/min)

0.100

0.050

0.000
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Total Chymotrypsin Concentration (mg/ml)

Using the burst heights of the PNPA

hydrolysis assay and the extinction coefficient of PNP it was possible to determine the percent
enzyme activity of the stock chymotrypsin solution. Total enzyme concentration was converted
to units of mM by dividing each value by 25 mg/mmol (the molar weight of bovine -
chymotrypsin). The burst heights were then converted from absorbance units to mM by dividing
by the calculated 405 of PNP (19.32 mM-1cm-1). The new burst heights were then plotted against
the mM enzyme concentrations (Figure 7). Taking the slope of the graph in Figure 7 and
multiplying by 100 resulted in a percent activity of 57.33 3.83 %.

Figure 5: The deacylation rates of chymotrypsin catalyzed

PNPA hydrolysis as a function of total enzyme concentration.
The graph shows a linear relationship of deacylation rate to
enzyme concentration. Data points are an average of three
repeated experiments.
 Figure 6: The burst heights of the PNPA hydrolysis assay as
a function of total enzyme concentration. The graph shows a
linear relationship of burst height to enzyme concentration.
Data points are an average of three repeated experiments.

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

Total Chymotrypsin Concentration (mM)

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

Figure 7: The dependence of burst height (mM) on

total chymotrypsin concentration (mM). The slope of
the best fit line through the origin corresponds to the
proportion of enzyme activity out of 1.