Kinetic studies of pepsin active site model compound and porcine pepsin

Article abstract of DOI:10.1002/1099-1395(200102)14:2<103::AID-POC342>3.0.CO;2-6

The kinetic parameters for the hydrolysis of the heptapeptide Pro-Thr-Glu-Phe-(4-NO₂)Phe-Arg-Leu by the pepsin model compound tetrabutylammonium monosalt of m-aminobenzoic acid diamide of fumaric acid (TBA m-FUM) and porcine pepsin were determined using a spectrophotometric technique. According to the AS* values obtained, in the transition state the inner motion in the TBA m-FUM-heptapeptide complex is more restricted than that in the pepsin-heptapeptide complex. The model compound TBA m-FUM can cause a cleavage of the Phe -(4-NO₂)Phe bond in the substrate molecules following a mechanism similar as that suggested for pepsin. but its catalytic activity is much lower. Copyright

Full text of DOI:10.1002/1099-1395(200102)14:2<103::AID-POC342>3.0.CO;2-6

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2001; 14: 103–108
Kinetic studies of pepsin active site model compound and
porcine pepsin
Bogdan Swoboda,1 Maria Bertowska-Brzezinska,1 Grzegorz Schroeder,1 Bogumil Brzezinski1* and
Georg Zundel2
1Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, PL-60780 Poznan, Poland
2Institute of Physical Chemistry, University of Munich, Theresienstr. 41, D-80333 Munich, Germany
Received 7 July 1999; revised 30 November 2000; accepted 1 November 2000
ABSTRACT: The kinetic parameters for the hydrolysis of the heptapeptide Pro–Thr–Glu–Phe-(4-NO2)Phe–Arg–Leu
by the pepsin model compound tetrabutylammonium monosalt of m-aminobenzoic acid diamide of fumaric acid
(TBA m-FUM) and porcine pepsin were determined using a spectrophotometric technique. According to the DS≠
values obtained, in the transition state the inner motion in the TBA m-FUM–heptapeptide complex is more restricted
than that in the pepsin–heptapeptide complex. The model compound TBA m-FUM can cause a cleavage of the Phe—
(4-NO2)Phe bond in the substrate molecules following a mechanism similar as that suggested for pepsin, but its
catalytic activity is much lower. Copyright 2001 John Wiley & Sons, Ltd.
KEYWORDS: pepsin model compounds; kinetics; UV spectra; peptide bond hydrolysis
INTRODUCTION
Recently, monosalts of o-, m- and p-aminobenzoic acid
diamides have been synthesized as model compounds
resembling the active site of pepsin.9 These compounds
include two carboxylic groups with a different distance
between them. Three of the model compounds showed
hydrolytic activity with respect to oxindole, while
tetrabutylammonium monosalt of m-aminobenzoic acid
diamide of fumaric acid (TBA m-FUM) also caused
cleavage of the heptapeptide Pro–Thr–Glu–Phe–(4-
NO2)Phe–Arg–Leu.9 The aim of this work was to study
the kinetics of this reaction. For the sake of comparison,
the reaction with pepsin was also investigated under
comparable experimental conditions.
The activity of pepsin has been assayed by hydrolysis of
naturally occurring proteins such as hemoglobin.1 How-
ever, many bonds in hemoglobin molecules have been
cleaved simultaneously and therefore this compound
appeared unsuitable for mechanistic studies.2 Conse-
quently, other substrates, i.e. dipeptides and polypep-
tides, were used in investigations of the catalytic action of
enzymes.3–5 The kinetic data for the hydrolysis of a series
of peptide substrates have revealed that the Phe—Phe and
Phe—(4-NO2)Phe bonds were hydrolysed more rapidly
than the other peptide bonds,5 while the reaction rate was
markedly affected by the structure of residues on both
sites of the scissile bond. The heptapeptide Pro–The–
Glu–Phe–(4-NO2)Phe–Arg–Leu was designed as one of
the best substrates for detailed studies on the stereo-
chemistry and intermolecular forces in the active site of a
number of the aspartic proteinases.6 By x-ray investiga-
tion it has been shown that an active site fissure of porcine
pepsin can be occupied by a heptapeptide.7 Such a result
was previously predicted by theoretical calculations.8
Analysis of heptapeptide hydrolysis products indicated
the formation of two peptides, Pro–Thr–Glu–Phe and (4-
NO2)Phe–Arg–Leu).6 It has been found that the kinetics
of the peptide bond splitting can be observed by a
spectrophotometric method involving measurement of
the change in absorbance at 300 nm.5,6,9,10
EXPERIMENTAL
Porcine pepsin and substrate
Pepsin was obtained from Aldrich-Chemie. The heptapep-
tide Pro–Thr–Glu–Phe–(4-NO2)Phe–Arg–Leu and isova-
lerylpepstatin were purchased from Bachem. All these
compounds were used as received. The activity of pepsin
was determined by titration of the enzyme against a
solution of isovalerylpepstatin as described by Dunn et al.6
Synthesis of tetrabutylammonium monosalt of
m-aminobenzoic acid diamide of fumaric acid
(TBA m-FUM)
*Correspondence to: B. Brzezinski, Faculty of Chemistry, A.
Mickiewicz University, Grunwaldzka 6, PL-60780 Poznan, Poland.
E-mail: bbrzez@mam.amu.edu.pl
Synthesis of m-FUM. m-FUM (Fig. 1) was prepared
from fumaric acid and m-aminobenzoic acid. Fumaric
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B. SWOBODA ET AL.
Figure 1. Structure of m-aminobenzoic acid diamide of
fumaric acid (m-FUM)
acid (0.01 mol) and oxalyl chloride (0.05 mol) were
stirred and refluxed for 24 h in chloroform. Fumaryl
chloride was separated, diluted in 50 cm3 of dioxane and
stirred with m-aminobenzoic acid for 24 h. Dioxane was
evaporated under reduced pressure. The m-FUM obtained
was filtered, washed with water and dried. The purity was
confirmed by elemental analysis: C 60.9 (61.0), H 4.1
(4.0), N 7.9 (7.9)%. The compound melted with
carbonization at 300°C. The reaction yield was 78%.
The 1H NMR spectrum of m-FUM shows four
aromatic signals at 7.75 (CH 6,6'), 7.51 (CH 5,5'), 7.96
(CH 4,4') and 8.42 ppm (CH 2,2'). The signal of CH 2,2'
is shifted owing to the closeness of carboxylic and amide
groups. The two acidic protons are found at 12.5 ppm.
Protons, which are attached to the double bonded carbons
and to the amide groups (NH), have signals at 7.2 and
10.93 ppm, respectively.
Figure 2. Change in UV absorbance of the heptapeptide
Pro±Thr±Glu±Phe±(4-NO2)Phe±Arg±Leu. Temperature,
293 K; solvent, hydrochloric acid (0.001 M), time interval,
10 s; heptapeptide concentration, 105 mM, pepsin concen-
tration, 23 nM
pepsin). The progress of the substrate hydrolysis by TBA
m-FUM or porcine pepsin was monitored by recording
the change in absorbance at 300 nm versus time (Figs 2
and 3). The kinetic runs were carried out using a stopped-
flow spectrophotometer (Applied Photophysics) with the
cell block termostated to Æ0.1°C.
The least-squares procedure was applied for computa-
tion of the initial reaction rates (V) with at least seven
values of the substrate concentration ([S]) at each
constant concentration of the enzyme model compound
or enzyme ([E]). In all cases, a hyperbolic V vs [S]
dependence was obtained according to the Michaelis–
Menten equation. The maximum velocity of the hepta-
peptide hydrolysis (VMAX) and the Michaelis–Menten
constant (KM) were derived from a non-linear regression
analysis of the experimental data and a linear regression
Synthesis of tetrabutylammonium monosalt of m-
FUM (TBA m-FUM).
A
solution of 8.80 mg
(2.5 Â 10 5 mol) of m-FUM in 5 cm3 of ethanol was
added to 0.025 cm3 of 1 m tetrabutylammonium hydro-
xide in methanol. The alcohol was evaporated under
reduced pressure and the m-FUM monosalt was dissolved
in 25 cm3 of water. From this stock solution we prepared
dilute solutions for further studies.
Mass spectra
Low-resolution liquid secondary ion mass spectrometry
(LSIMS) of the reaction products after completed
hydrolysis of the heptapetide Pro–Thr–Glu–Phe–(4-
NO2)Phe–Arg–Leu with TBA m-FUM was performed
on an AMD 604 two-sector mass spectrometer (AMD
Intectra, Germany) of BE geometry. m-Nitrobenzyl
alcohol (NBA) was used as a matrix.
Determination of kinetic parameters
Solutions of the heptapeptide Pro–Thr–Glu–Phe–(4-
NO2)Phe–Arg–Leu of concentration ranging from 5.2
to 209 mm were prepared in water (in the case of TBA m-
Figure 3. Change in UV absorbance of the heptapeptide
Pro±Thr±Glu±Phe±(4-NO2)Phe±Arg±Leu. Temperature, 293 K;
solvent, water; time interval, 10 s; heptapeptide concentra-
tion, 105 mM; TBA m-FUM concentration, 99 mM
3
FUM) or in 10 m hydrochloric acid (in the case of
Copyright 2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 103–108

KINETIC STUDIES OF PEPSINS
105
Figure 4. LSIMS of the product mixture obtained having completed hydrolysis of the heptapeptide Pro±Thr±Glu±Phe±(4-
NO2)Phe±Arg±Leu by TBA m-FUM
analysis of the Lineweaver–Burk, Hanes and Eadie–
Hofstee plots.11,12 The catalytic constants, kcat = VMAX
conclusion drawn from UV and IR data that the only bond
cleaved is the Phe–(4-NO2)Phe bond.9 The formation of
the tripeptide and tertrapeptide is well evidenced by the
peaks: m/z 480 for (4-NO2)Phe–Arg–Leu and m/z 493 for
Pro–Thr–Glu–Phe. The m/z 353 peak can be assigned to
the m-FUM anion.
The kinetic parameters of the Michaelis–Menten
equation are given in Table 1. The value of kcat./KM for
pepsin is considerably higher than that for TBA m-FUM,
whereas the affinity of TBA m-FUM to the substrate is
twice higher. The catalytic activity (kcat) of TBA m-FUM
in the hydrolysis of the substrate molecules is about three
orders of magnitude lower than that of porcine pepsin.
This difference in kcat values may be caused by the
specific interactions existing between various parts of the
heptapeptide and pepsin but not between the substrate
and TBA m-FUM.
/
[E], were calculated using the experimentally obtained
VMAX values and the appropriate concentration of TBA
m-FUM or porcine pepsin. The pseudo-first-order rate
constants (kobs) were found from the time dependence of
absorbance according to the equation kobs (1/t) ln
(A? Ao)/(A? At), where A0 initial absorbance,
At = absorbance at time t and A? = absorbance at infinite
time. The activation entropy (DS≠), enthalpy (DH≠) and
free enthalpy (DG≠) were evaluated from the Eyring’s
equation by a linear least-squares fit of ln kobs vs 1/T.
RESULTS AND DISCUSSION
The hydrolysis of the heptapeptide Pro–Thr–Glu–Phe–
(4-NO2)Phe–Arg–Leu as a substrate by TBA m-FUM
yields two peptides, which were identified by mass
spectrometry technique. An example of the mass
spectrum of the product mixture taken having completed
the hydrolysis of the heptapeptide by m-FUM is given in
Fig. 4. Examination of this mass spectrum confirms the
The pseudo-first-order rate constants for the reactions
of heptapetide with porcine pepsin and TBA m-FUM are
given in Tables 2 and 3, respectively. In the case of
pepsin, with increasing temperature the kobs. values
increase continuously, whereas in the case of TBA m-
FUM, the kobs. values increase only up to 308 K. Because
Table 1. Kinetic parameters for the hydrolysis of the heptapetide Pro±Thr±Glu±Phe±(4-NO2)Phe±Arg±Leu by TBA m-FUM and
porcine pepsin at 293 K
1
1
1
Substance
[E] (mM)
Average KM (mM)
Average VMAX (mMs
)
kcat. (s
)
kcat./KM (mM 1s
)
3
3
TBA m-FUM
Porcine pepsin
99
29 Â 10
61 Æ 8
0.25 Æ 0.01
0.31 Æ 0.02
(2.5 Æ 0.1) Â 10
10.7 Æ 0.7
(46 Æ 6) Â 10
95 Æ 8
3
113 Æ 16
Copyright 2001 John Wiley & Sons, Ltd.
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106
B. SWOBODA ET AL.
k
Copyright 2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 103–108

KINETIC STUDIES OF PEPSINS
107
Table 3. Pseudo-®rst order rate constants kobsfor the hydrolysis of the heptapeptide Pro±Thr±Glu±Phe±(4-NO2)Phe±Arg±Leu by
TBA m-FUM in water, with TBA m-FUM concentrations of 49.71±198.87 mM as indicated
1
kobs (s
)
Temperature
(K)
49.71mM
99.44mM
148.31mM
198.87mM
288
298
308
318
0.0032 Æ 0.0001
0.0055 Æ 0.0001
0.0072 Æ 0.0001
0.0048 Æ 0.0001
0.0059 Æ 0.0001
0.0080 Æ 0.0002
0.0140 Æ 0.0002
0.0092 Æ 0.0001
0.0094 Æ 0.0001
0.0127 Æ 0.0001
0.0260 Æ 0.0002
0.0141 Æ 0.0002
0.0113 Æ 0.0002
0.0181 Æ 0.0003
0.0267 Æ 0.0004
0.0159 Æ 0.0003
negative value of the (DS≠) explains the higher (DG≠)
value for the reaction of heptapetide with TBA m-FUM,
although the activation enthalpy is clearly lower.
Previous work on the mechanism of the pepsin activity
Table 4. Activation parameters for the hydrolysis of the
heptapeptide Pro±Thr±Glu±Phe±(4-NO2)Phe±Arg±Leu by
TBA m-FUM and porcine pepsin
Activation
Activation
Free
enthalpy, DH≠ entropy, DS≠ enthalpy, DG≠
suggested that the H2O or OH species were located in
9,13–15
1
1
1
Substance
(kJ mol
)
(J mol 1K
)
(kJ mol )
the active centre of pepsin
and, consequently, a
base mechanism of hydrolysis was proposed. Recently,
we have also assumed this type of mechanism for the
cleavage of heptapeptide Pro–Thr–Glu–Phe–(4-
NO2)Phe–Arg–Leu) by TBA m-FUM.9 The above-
presented kinetic data for the heptapeptide hydrolysis
by both pepsin and TBA m-FUM support this assump-
tion.
The mechanism of the TBA m-FUM activity is shown
in Fig. 5. The water molecule bound between the
carboxylic and carboxylate groups of TBA m-FUM
becomes distorted when approached by the heptapeptide
molecule. The carbonyl group of the heptapeptide forms
a hydrogen bond with the carboxylic group of TBA m-
FUM while the negative partial charge of the O atom of
water attacks the electrophilic carbon atom of the peptide
bond. The formation of internal hydrogen bonds is easily
visible in the FTIR spectrum.9 The hydroxyl ion resulting
from the split H2O molecule adds to the carbon atom of
the peptide bond of heptapetide. Simultaneously, the
TBA m-FUM
Porcine pepsin
31 Æ 3
53 Æ 5
100 Æ 7
36 Æ 2
61 Æ 3
41 Æ 5
of the possible decomposition of the model compound
(TBA m-FUM) at higher temperatures, the Michaelis–
Menten kinetic parameters were obtained at 293 K (see
Table 1). Changes in pH conditions of the hydrolysis
reactions may be a further factor contributing to the
differences in the observed kinetics.
The activation parameters are listed in Table 4. The
entropy of activation (DS≠) reflects the changes in the
number and kind of the degrees of freedom if the reagents
form an active complex. When the complex movement in
the transition state is limited or impeded, (DS≠) decreases.
Consequently, the possibility of inner motion in the TBA
m-FUM–heptapeptide complex should be more restricted
than that of the pepsin–heptapeptide complex. The
Figure 5. Suggested mechanism of the hydrolysis of the heptapeptide Pro±Thr±Glu±Phe±(4-NO2) Phe±Arg±Leu by TBA m-FUM
Copyright 2001 John Wiley & Sons, Ltd. J. Phys. Org. Chem. 2001; 14: 103–108

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B. SWOBODA ET AL.
amide bond is broken and the proton adds to the nitrogen
atom.
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Conclusions
The steric arrangement of carboxylic and carboxylate
groups in the tetrabutylammonium monosalt of m-
aminobenzoic acid diamide of fumaric acid (TBA m-
FUM) has proved beneficial for catalytic hydrolysis of
the heptapeptide Pro–Thr–Glu–Phe–(4-NO2)Phe–Arg–
Leu in water. The mechanism of cleavage of the Phe—
(4-NO2)Phe bond by TBA m-FUM is similar to that of
pepsin. However, the catalytic activity of TBA m-FUM,
indicated by the kcat values, is much lower than that of
pepsin, although the affinity of TBA m-FUM to the
heptapeptide is higher than the affinity of pepsin. As
follows from the activation parameters, the inner motion
in the complex of the TBA m-FUM–heptapeptide in the
transition state is more restricted than that in the pepsin–
heptapeptide complex.
15. Zundel G, Iliadis G, Brzezinski B. FEBS Lett. 1994; 325: 315–317.
Copyright 2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 103–108