A re-examination of two linear pentapeptides claimed to be serine protease mimics

Article abstract of DOI:10.1021/ja9616140

The synthesis, characterization, and kinetic study of two linear pentapeptides L-threonyl-L-alanyl-L-seryl-L-histidyl-L-aspartic acid (TASHD) and L-seryl-γ-aminobutyryl-L-histidyl-γ-aminobutyryl-L-aspartic acid (Ser-Gaba-His-Gaba-Asp) that were previously

Full text of DOI:10.1021/ja9616140

J. Am. Chem. Soc. 1996, 118, 8787-8790
8787
A Re-examination of Two Linear Pentapeptides Claimed To Be
Serine Protease Mimics
Anthony M. Vandersteen and Kim D. Janda*
Contribution from The Scripps Research Institute, Departments of Molecular Biology and
Chemistry, The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road,
La Jolla, California 92037
ReceiVed May 14, 1996X
Abstract: The synthesis, characterization, and kinetic study of two linear pentapeptides L-threonyl-L-alanyl-L-seryl-
L-histidyl-L-aspartic acid (TASHD) and L-seryl-γ-aminobutyryl-L-histidyl-γ-aminobutyryl-L-aspartic acid (Ser-Gaba-
His-Gaba-Asp) that were previously reported by J. C. Sheehan et al. (Cruickshank, P.; Sheehan, J. C. J. Am. Chem.
Soc. 1963, 86, 2070-2071 and Sheehan, J. C.; Bennett, G. B.; Schneider, J. A. J. Am. Chem. Soc. 1966, 88, 3455-
3456) to be esterolytic catalysts modeling the active site of chymotrypsin is described. In contrast to these two
reports, we observed no catalysis of ester hydrolysis for substrates p-nitrophenyl acetate (p-NPA), N-methoxycarbonyl-
L-and-D-phenylalanine-p-nitrophenyl esters using the original conditions reported. In order to probe our conflicting
results, accurate measurement of second order rate constants for the hydrolysis of each of these three p-nitrophenyl
esters was carried out. The second order rate constants for these hydrolysis reactions were an order of magnitude
lower than those originally reported for peptide Ser-Gaba-His-Gaba-Asp and three times lower than those reported
for peptide TASHD. A very small difference in the rates of hydrolysis of N-methoxycarbonyl-L-and-D-phenylalanine-
p-nitrophenyl esters in the presence of peptides TASHD and Ser-Gaba-His-Gaba-Asp was observed. Kinetic studies
of the hydrolysis of p-NPA in the presence of peptide TASHD using a variety of concentrations of substrate and
peptide are reported. A series of measurements of initial rates of hydrolysis of p-NPA in the presence of peptide
TASHD provided no evidence for saturation kinetics. Treatment of peptide TASHD with the serine-protease inhibitor
diisopropylfluorophosphate (DFP) produced no retardation in the initial rates of hydrolysis of p-NPA. The second
order rate constants for the two peptides fell on the Brønsted line for a variety of substituted imidazoles from the
literature. Our interpretation of these findings is that the peptides are behaving as simple imidazole catalysts for the
hydrolysis of p-NPA and that there is no evidence for the involvement of the serine residue side chain during catalysis
or any substrate binding by these peptides.
Introduction
and co-workers were among the first attempts to mimic the
active site of an enzyme using a chemically synthesized model
peptide.7,8,10
The serine-protease family of enzymes is integral to the
physiological processes of food digestion, cleavage of signal
peptides, and control of blood pressure and blood clotting.1 They
constitute one of the most intensively studied families of
enzymes, with a large number of biochemical and chemical
reports providing a better understanding of their mechanism and
the characteristics of enzyme catalysis in general.2-4
The catalytic triad of chymotrypsin (histidine, serine, and
aspartic acid), has been incorporated into linear peptides,5-8
cyclic peptides,9-11 polymers of amino acids,12-14 and small
organic compound models.15,16 Three reports by J. C. Sheehan
The discovery of novel pharmaceutical lead compounds,
synthetic receptors, and solid state materials has recently been
reported using “combinatorial” approaches.17,18 We and oth-
ers19,20 have been interested in applying combinatorial chemistry
to the area of de noVo peptide catalyst design or what we term
“combizymes”. As a starting point for such studies we decided
to reinvestigate the two linear pentapeptides: L-threonyl-L-
alanyl-L-seryl-L-histidyl-L-aspartic acid (TASHD)7,8 and L-seryl-
γ-aminobutyryl-L-histidyl-γ-aminobutyryl-L-aspartic acid (Ser-
Gaba-His-Gaba-Asp).7 These peptides had been reported by
Sheehan et al. to catalyze the hydrolysis of p-nitrophenyl acetate
(p-NPA) with second order rate constants 3 to 25 times greater
* To whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, September 1, 1996.
(1) Branden, C.; Tooze, J. Introduction to protein structure; Garland
Publishing Inc.: 1991.
(2) Jencks, W. P. Catalysis in chemistry and enzymology; Dover
Publications Inc.: 1987.
(11) Schultz, R. M.; Huff, J. P.; Anagnostaras, P.; Olsher, U.; Blout, E.
R. Int. J. Peptide Protein Res. 1982, 19, 454-469.
(12) Katchalski, E.; Fasman, G. D.; Simons, E.; Blout, E. R.; Gurd, F.
R. N.; Koltun, W. L. Arch. Biochem. Biophysics 1960, 88, 361-365.
(13) Nishi, N. Peptide Chemistry 1978, 151-156.
(14) Mutter, M. Angew. Chem., Int. Ed. Engl. 1985, 24, 639-653.
(15) Mallick, I. M.; D’Souza, V. T.; Yamaguchi, M.; Lee, J.; Chalabi,
P.; Gadwood, R. C.; Bender, M. L. J. Am. Chem. Soc. 1984, 106, 7252-
7254.
(3) Fersht, A. Enzyme structure and mechanism; W. H. Freeman and
Co.: 1985.
(4) Kraut, J. Ann. ReV. Biochem. 1977, 46, 331-358.
(5) Vorherr, T.; Altmann, K.-H.; Mutter, M. HelV. Chim. Acta 1986, 69,
410-414.
(6) Photaki, I.; Sakarellou-Daitsiotou, M. J. Chem. Soc., Perkin Trans.
I 1976, 589-591.
(16) Cram, D. J.; Lam, P. Y.-S.; Ho, S. P. J. Am. Chem. Soc. 1986, 108,
839-841.
(7) Sheehan, J. C.; Bennett, G. B.; Schneider, J. A. J. Am. Chem. Soc.
1966, 88, 3455-3456.
(17) Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10779-10785.
(18) Hsieh-Wilson, L. C.; Xiang, X.-D.; Schultz, P. G. Acc. Chem. Res.
1996, 29, 164-170.
(8) Cruickshank, P.; Sheehan, J. C. J. Am. Chem. Soc. 1963, 86, 2070-
2071.
(9) Kopple, K. D.; Nitecki, D. E. J. Am. Chem. Soc. 1962, 84, 4457-
4464.
(10) Sheehan, J. C.; Mcgregor, D. N. J. Am. Chem. Soc. 1962, 84, 3000-
3005.
(19) Menger, F. M.; Eliseev, A. V.; Migulin, V. A. J. Org. Chem. 1995,
60, 6666-6667.
(20) Perez-Paya, E.; Houghten, R. A.; Blondelle, S. E. Peptide Research
1994, 7, 286-288.
S0002-7863(96)01614-9 CCC: $12.00 © 1996 American Chemical Society

8788 J. Am. Chem. Soc., Vol. 118, No. 37, 1996
Vandersteen and Janda
Table 2. Second Order Rate Constants (L mol-1 s-1)a for the
Hydrolysis of N-Methoxycarbonyl-L-and-D-phenylalanine-p-nitro-
phenyl Esters
k2 (L mol-1 s-1
L-substrate
)
k2 (L mol-1 s-1
)
D-substrate
catalyst
peptide TASHD
peptide Ser-Gaba-His-Gaba-Asp
0.33
0.25
0.23
0.12
a Second order rate constants were measured by varying peptide
concentrations between 0.57 and 2.83 mM with a substrate concentra-
tion of 28.2 µM in 3.2% (by volume) of dioxane in buffer (0.2 M
phosphate pH 7.8) at 28.0 °C ( 0.5 °C.
Figure 1. A plot of k1 - kw (min-1) vs peptide concentration (mM)
for peptide TASHD and substrate p-NPA. Reactions were carried out
in 3.2% by volume of 1,4-dioxane in 0.2 M phosphate buffer at pH
7.7, 25.5° ( 0.5 °C using a p-NPA concentration of 28.2 µM and
varying peptide TASHD concentrations. The plot shows two measure-
ments of k1 - kw at each concentration of peptide and the line is fitted
to the average of the two values.
Table 1. Second Order Rate Constants (L mol-1 s-1)a for the
Hydrolysis of p-Nitrophenyl Acetate
catalyst
peptide TASHD
peptide Ser-Gaba-His-Gaba-Asp
k2 (L mol-1 s-1
)
Figure 2. A Brønsted plot for a series of substituted imidazoles23 and
peptides (I) and (II) (a) 4(5)-bromoimidazole, (b) benzimidazole, (c)
4(5)-hydroxymethylimidazole, (d) imidazole, (e) 4(5)-methylimidazole,
(f) peptide (I), and (g) peptide (II).
0.29
0.14
a Second order rate constants were measured by varying peptide
concentrations between 0.57 and 2.83 mM with a substrate concentra-
tion of 28.2 µM in 3.2% (by volume) of dioxane in buffer (0.2 M
phosphate pH 7.8) at 28.0 °C ( 0.5 °C.
than imidazole and L-histidine, respectively, to show catalytic
stereoselectivity and to be subject to inhibition by the serine
protease inhibitor DFP. The conclusion from these findings
was that polyfunctional effects associated with enzyme active
sites might be operative in these two peptides. We investigated
each of these claims and also sought to determine if any
peptide-substrate complex and hence saturation kinetics could
be observed.
Results
Kinetic studies for the hydrolysis of p-NPA and N-methoxy-
carbonyl-L-and-D-phenylalanine-p-nitrophenyl esters in the pres-
ence of peptide TASHD were carried out using the conditions
originally reported by Sheehan et al.8 No rate acceleration was
observed under these conditions. A second set of conditions
reported by Sheehan et al.7,10 was used to measure the catalysis
produced by peptides TASHD and Ser-Gaba-His-Gaba-Asp; the
small rate acceleration over background which was observed
could not be accurately quantified under these conditions.
In a second series of experiments a higher concentration of
peptide was used. A plot of k1 - kw (min-1) vs peptide TASHD
concentration (mM) for the hydrolysis of p-nitrophenyl acetate
is shown in Figure 1. The second order rate constants measured
for the hydrolysis of p-NPA in the presence of TASHD and
Ser-Gaba-His-Gaba-Asp are reported in Table 1. The second
order rate constants measured for the hydrolysis of N-meth-
oxycarbonyl-L-and-D-phenylalanine-p-nitrophenyl esters in the
presence of TASHD and Ser-Gaba-His-Gaba-Asp are reported
in Table 2.
Figure 3. A plot of initial rate of reaction V (mM min-1) vs p-NPA
concentration [S] (mM) for peptide TASHD. Reactions were carried
out in 5% by volume of 1,4-dioxane in 0.1 M phosphate buffer at pH
7.6, 35.0° ( 0.5 °C using a peptide concentration of 500 µM and
varying p-NPA concentrations. The plot shows the mean value of two
measurements of the initial rate at each substrate concentration.
histidyl-L-aspartic acid (TACHD) proved fruitless. We observed
a linear increase in the initial rates of reaction with increasing
substrate concentration using a number of different concentra-
tions of peptide. A plot of the initial rates of hydrolysis of
p-NPA in the presence of 500 µM peptide TASHD and the
background rates is shown in Figure 3.
The effect of a serine specific protease inhibitor diisopropy-
lfluorophosphate (DFP) on the reaction rate was measured.
Despite a large molar excess of DFP we saw no decrease in the
initial rates of reaction relative to the control experiments.
Discussion
The original data reported by Sheehan et al.7,10 is summarized
in Tables 3 and 4. It is evident after comparing the data in
Attempts to observe saturation type kinetics with peptide
TASHD and p-NPA substrate using conditions previously
reported by Lowe et al.21 in their study of the cysteine protease
analog of peptide TASHD, L-threonyl-L-alanyl-L-cysteinyl-L-
(21) Photaki, I.; Bardakos, V.; Lake, A. W.; Lowe, G. J. Chem. Soc.
(C) 1968, 1860-1864.

Two Linear Pentapeptides
J. Am. Chem. Soc., Vol. 118, No. 37, 1996 8789
Table 3. Reported Second Order Rate Constants (L mol-1 s-1) for
the Hydrolysis of p-Nitrophenyl Acetate7,8,24
Brønsted analysis for both TASHD and Ser-Gaba-His-Gaba-
Asp (Vide supra) has led us to the conclusion that the histidine
residue of these peptides is acting as a simple chemical catalyst
(either nucleophilic or general base catalysis of the magnitude
expected for imidazoles of similar pKa). This can be contrasted
with the role of histidine in activating the serine hydroxyl group
for nucleophilic catalysis in the active site of the serine protease
enzymes. In addition the small difference in second order rate
constants for the hydrolysis of N-methoxycarbonyl-L-and-D-
phenylalanine-p-nitrophenyl esters by peptides (TASHD) and
(Ser-Gaba-His-Gaba-Asp) does not provide evidence for ste-
reoselective substrate recognition by the peptides.
None of the characteristics of enzyme catalysis, as seen with
chymotrypsin such as substrate recognition/binding, formation
of an acyl-enzyme intermediate or rate acceleration/transition
state stabilization beyond that produced by substituted imida-
zoles is produced by these two peptides.
catalyst
peptide TASHD
peptide Ser-Gaba-His-Gaba-Asp
peptide TASHD + DFP
imidazole
k2 (L mol-1 s-1
)
1.53
2.45
0.80
0.33 (ref 7) 0.58 (ref 24)
Table 4. Reported Second Order Rate Constants (L mol-1 s-1) for
the Hydrolysis of N-Methoxycarbonyl-L-and-D-phenylalanine-p-
nitrophenyl Esters7
k2 (L mol-1 s-1
L-substrate
)
k2 (L mol-1 s-1
)
D-substrate
catalyst
peptide TASHD
peptide Ser-Gaba-His-Gaba-Asp
imidazole
1.03
2.58
1.00
0.53
1.87
1.00
Tables 1 and 3 that the second order rate constants for the
hydrolysis of p-NPA are significantly lower than those originally
reported and are of a similar magnitude as those reported for
other histidine-containing peptide enzyme models.6,11,13 In order
to probe what interactions may be taking place between the
peptide and substrate during the reaction we constructed a
Brønsted plot using available second order rate constants for
the hydrolysis of p-NPA. A discussion of general base catalysis
and its relationship to enzymatic hydrolysis has been reported
for a variety of bases22 and substituted imidazoles.23 Detailed
studies of the catalysis of ester hydrolysis for a variety of
substrates by imidazole has also been reported.24,25 A Brønsted
plot for the second order rate constants of the hydrolysis of
p-NPA by a variety of four (5)-substituted imidazoles23 together
with peptides TASHD and Ser-Gaba-His-Gaba-Asp is shown
in Figure 2. It is clear that the second order rate constants for
these peptides fall closely on the Brønsted line of gradient 0.64.
Based on this plot we contend that peptides TASHD and Ser-
Gaba-His-Gaba-Asp are acting as substituted imidazoles under
these experimental conditions and are not exhibiting any
enhanced catalytic acceleration connected with the presence of
the “catalytic triad” of chymotrypsin.
With respect to the hydrolysis of N-methoxycarbonyl-L-and-
D-phenylalanine-p-nitrophenyl esters in the presence of peptides
TASHD and Ser-Gaba-His-Gaba-Asp, we observed significantly
lower second order rate constants than reported by Sheehan et
al.7 (Tables 2 and 4).
The difference between the second order rate constants for
the L and D substrates is five times smaller than originally
reported. Such an insignificant difference in rate constants led
us to conclude that these peptide enzyme models were not
displaying significant stereoselectivity, especially in comparison
to the exquisite stereoselectivity displayed by enzymes.3
Sheehan and co-workers originally observed a 48% decrease
in the catalytic coefficient after treatment of the peptide
(TASHD) with DFP.7 Despite a large molar excess of DFP
we saw no decrease in the initial rates of reaction relative to
the control experiments and thus concluded that the serine
residue was not involved in the catalytic mechanism.
Experimental Section
Materials. Fully protected amino acid building blocks and deriva-
tized Wang resins were purchased from Novabiochem. N,N-Dimeth-
ylformamide, N-methylpyrollidone, isopropyl alcohol, and methylene
chloride were used as supplied by Baxter. Diisopropylcarbodiimide,
1-hydroxybenzotriazole, piperidine, ethane dithiol, and trifluoroacetic
acid were used as supplied by the Aldrich Chemical Company.
p-Nitrophenyl acetate was purchased from the Sigma chemical company
and was used without further purification, 1,4-dioxane of the purest
grade available was used during kinetic runs without futher purification.
Volumetric standard solutions of 0.1 N KOH and pH calibration
standard solutions of pH 4 and 7 were purchased from Fisher scientific.
Methods. Peptides TASHD and Ser-Gaba-His-Gaba-Asp were
synthesized by manual solid-phase synthesis using N-terminal fluorenyl-
methoxycarbonyl (FMOC) protection and acid labile side chain
protection according to published methods.26 The peptides were
purified by preparative reversed phase HPLC and were fully character-
ized. Physical data for peptide TASHD matched Sheehan’s data
closely; however, we measured a different melting point and optical
rotation from the reported values for peptide Ser-Gaba-His-Gaba-Asp.
It should be noted that the original synthetic work was carried out using
the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride coupling reagent27 which has recently been reported to lead
to significant racemization during amino acid coupling reactions.28
TASHD was purified by reversed phase HPLC using a Waters prep
LC4000 and Waters 1000 prepak column module eluting with 99.9%
H2O 0.1% TFA. Peptide Ser-Gaba-His-Gaba-Asp was purified using
a Rainin HPXL and a Vydac C18 90Å semipreparative column eluting
with a gradient 0-40% acetonitrile in 0.1%TFA/water. Analytical
HPLC was carried out on a Hitachi L6200A instrument with a reversed
phase Vydac C18 column. Detection of the peptides during preparative
and analytical HPLC was carried out at 210 nm.
All pH titrations were carried out according to reported methods29
in duplicate with a Corning general purpose electrode and an Aldrich
combination ultrathin long-stem electrode calibrated between pH 4 and
7 on an Orion pH/ISE meter. The pKa of the histidine residue for each
peptide was determined by direct potentiometric titration of a 0.01 N
aqueous peptide solution with standardized 0.1 N solution of potassium
hydroxide at 25 ( 0.5 °C with stirring by bubbling nitrogen gas through
the solution. The pKa value was determined as the point of inflection
in the region of imidazole ionization.12 No pKa values were reported
in the original papers.7,8
Melting points were measured on a Fisher-Johns melting point
apparatus and are uncorrected. Optical rotations were measured with
Conclusion
The initial rates of reaction in the presence of peptide TASHD
and the results of the DFP experiment coupled with the above
(26) Atherton, E.; Sheppard, R. C. Solid phase peptide synthesis, a
practical approach; IRL Press, 1989.
(22) Bender, M. L.; Turnquest, B. W. J. Am. Chem. Soc. 1957, 79, 1656-
1662.
(27) Sheehan, J. C.; Preston, J.; Cruickshank, P. A. J. Am. Chem. Soc.
1965, 87, 2492-2493.
(23) Bruice, T. C.; Schmir, G. L. J. Am. Chem. Soc. 1957, 79, 1663-
(28) Ho, G.-J.; Emerson, K. M.; Mathre, D. J.; Shuman, R. F.; Grabowski,
E. J. J. J. Org. Chem. 1995, 60, 3569-3570.
1667.
(24) Kirsch, J. F.; Jencks, W. P. J. Am. Chem. Soc. 1964, 86, 833-837.
(25) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1961, 1743-1750.
(29) Albert, A.; Serjeant, E. P. The determination of ionization constants,
a laboratory manual, 3rd ed; Chapman and Hall.

8790 J. Am. Chem. Soc., Vol. 118, No. 37, 1996
Vandersteen and Janda
a Perkin-Elmer 241 polarimeter. 1H-NMR spectra were recorded on a
Bruker AM300 spectrometer with tetramethylsilane as an internal
standard, and coupling constants are reported in Hertz. High resolution
fast atom bombardment (FAB) and electrospray ionization (ESI) mass
spectra were recorded by the Scripps Research Institute mass spec-
trometry facility.
Kinetic Experiments. The progress of the reactions were measured
by the release of the p-nitrophenolate ion at 405 nm in a quartz ELISA
plate with a Molecular Devices Spectramax 500 plate reader.30 A stock
peptide solution was prepared by dissolving 4-5 mg of peptide in 0.1
M phosphate buffer. A typical reaction volume was 200 µL. The
aqueous buffered peptide solution was transferred into an ELISA well
and prewarmed for at least 15 min. A solution of the substrate ester
in 1,4-dioxane was added, and the reaction monitoring was started
immediately using the SoftMax Pro software program. Values of initial
rates of reaction in OD min-1 were converted into µM min-1 of
p-nitrophenol by determining the conversion factor using standardized
p-nitrophenol solutions. The DFP solutions were prepared using
recommended safety procedures in a fume-hood. These experiments
were carried out in a disposable plastic ELISA plate.
of 1.24 mM. These conditions allowed reaction rates of both the peptide
and background to be accurately determined. The relationship seen
between peptide concentration and the initial rates of p-NPA hydrolysis
was linear.
In a further set of experiments, initial rates of p-NPA hydrolysis
were measured at peptide concentrations of 100 and 500 µM and p-NPA
concentrations between 1 and 4 mM. A linear relationship was
observed between the initial rates of reaction and substrate concentra-
tion. A plot of initial rate (µmol min-1) Vs [p-NPA] for 500 µM peptide
TASHD and the background rate is shown in Figure 3.
Inhibition studies with DFP were carried out by incubation of
solutions of peptide TASHD (343 µM in sodium phosphate buffer 0.15
M pH 7.61) with solutions of DFP (in acetonitrile with concentrations
ranging from 0.38 to 6.1 mM) for 15 min at room temperature. The
substrate p-NPA in dioxane (2.2 mM) was added and the initial rates
of reaction were observed at 25° ( 0.5 °C. The background rate
without added DFP or peptide was 5.31 µmol min-1, the rate with
peptide was 10.77 µmol min-1 and the rate with added peptide and
DFP was 9.80 µmol min-1
.
Analytical Data. Thr-Ala-Ser-His-Asp (TASHD).8 δ[1H 1:9 D2O:
H2O v/v, internal reference: 1,4 dioxane] 1.3 (3H, d, J ) 7.2), 1.4
(3H, d, J ) 8.1), 2.9 (2H, d, J ) 7.2), 3.2-3.4 (2H, m), 3.8 (2H, d, J
) 4.5), 3.9 (1H, d, J ) 7.2), 4.2 (1H, t, J ) 7.2), 4.4 (2H,m), 7.3 (1H,
s), 8.45-8.6 (4H,m), 8.55 (1H, s) 8.85 (2H, d, J ) 5.3) Analytical
HPLC Rt ) 5.6 min (eluent water/0.1% TFA). m/z FAB ms )
530.2208 [C20H31N7O10 requires 530.2211], RD(H2O) ) -4.3° [lit.
-8.9°], Tm ) 170 °C [lit. 177 °C], pKa ) 7.2 ( 0.1.
The reactions using p-NPA, N-methoxycarbonyl-L-and-D-phenyl-
alanine-p-nitrophenyl esters (concentration 28.2 µM) were followed
over at least five half-lives, and the pseudo-first order rate constants k1
were calculated by the semi-log method.2,7,12 The second order rate
-1
constant k2 (L mol min-1) was calculated according to eq 1.12
k2 ) (k1 - kw)/c
(1)
Amino acid analysis was carried out by the Scripps Research Institute
Core Facility and gave the following ratios of amino acids: Asp/His
(1): 0.99, 1.02 Ser (1): 0.98, 1.11 Ala (1): 1.0, 0.97 Thr (1): 0.94,
1.01.
where k1(min-1) ) measured pseudo-first order rate constant in the
presence of the catalyst, kw (min-1) is the background rate under
identical conditions in the absence of catalyst, and c (mol L-1) is the
molar concentration of uncharged histidine residues.
Determination of values for the pseudo-first order rate constants at
five different concentrations of each peptide between 0.57 and 2.83
mM in duplicate ensured reproducibility.2 The concentration of
uncharged histidine residues in solution was calculated using the
Henderson-Hasselbalch eq.2
Enzyme saturation kinetics are normally investigated by measuring
initial rates of reaction using a large excess of substrate over catalyst.
Lowe et al.21 had observed saturation kinetics during the hydrolysis of
p-NPA in the presence of the peptide TACHD with a Km ) 1.18 (
0.06 mM and a kcat ) 21 ( 0.2 × 10-3 s-1. A larger percentage of
dioxane and an increased temperature was required to ensure that the
substrate remained in solution. Thus reactions were carried out in 5%
(by volume) of dioxane in buffer (0.1 M phosphate pH 7.61) at 35.0 (
0.5 °C with a peptide concentration of 50 µM and seven substrate
concentrations ranging from 0.31 to 28.00 mM.21
Ser-Gaba-His-Gaba-Asp.7 δ[1H 1:9 D2O:H2O v/v, internal refer-
ence: 1,4 dioxane] 0.8 (8H, q, J ) 7.9,9.5), 1.65-1.9 (4H, m) 2.9
(2H, m), 3.1-3.3 (2H, m), 3.9-4.2 (2H, m), 4.15 (1H, t, J ) 4.7), 4.2
(2H, m), 7.4 (1H, s), 8.4 (1H, d, J ) 7.9) , 8.5 (1H,d, J ) 7.9), 8.58
(1H, s), 8.75 (2H, t, J ) 3.9). Analytical HPLC Rt ) 14.8 min (0 to
40% acetonitrile: 0.1% TFA/water over 40 min) m/z FAB ms )
550.2253 [C21H33N7O9Na requires 550.2237], RD (H2O) ) -33.5° [lit.
+25.6°], Tm ) 165-168 °C [lit. 280 °C dec], pKa ) 7.1 ( 0.1.
N-methoxycarbonyl-L-and-D-phenylalanine-p-nitrophenyl esters were
synthesized according to the reported procedure.31
L isomer δ[1H DMSO-d6] 3.2 (2H, t, J ) 8.1) 3.7 (3H, s), 4.8 (1H,
m), 5.25 (1H, d, J ) 9.7), 6.9-7.3 (7H, m), 8.2 (2H, m). m/z FAB ms
) 345.1099 [C17H16N2O6 requires 345.1087], RD (H2O) ) -8.9° [lit.
-9.6°], Tm ) 104 °C [lit. 103 °C]. D isomer m/z FAB ms ) 345.1097
[C17H16N2O6 requires 345.1087], RD (H2O) ) +10.9° [lit. +9.8°], Tm
) 104 °C [lit. 103 °C].
These experiements were carried out with peptide TASHD in
Acknowledgment. We would like to thank Dr. Arnold C.
Satterthwait and Dr. Edelmira Cabezas for assistance with the
solid-phase synthetic methodology, Professor Jack F. Kirsch for
critical reading of the manuscript, and Dr. Paul Wentworth, Jr.
for preparation of the manuscript and assistance with the kinetic
analysis. This work was supported in part by the Scripps
Research Institute and The Skaggs Institute for Chemical
Biology.
duplicate, and the data were analyzed by an initial rates analysis.2,21
A
slight rate enhancement was observed using these conditions (initial
rate of appearance of p-nitrophenolate in the presence of peptide
TASHD (µmol/min-1)/background rate (µmol/min-1) ≈ 1.1 ( 0.1);
however, it was too small to permit accurate determination of any
kinetic parameters, due to random experimental error.
Initial rates were next determined by increasing peptide concentra-
tions from between 100 and 500 µM at a fixed substrate concentration
JA9616140
(30) Eisenthal, R.; Danson, M. J. Enzyme Assays, a practical approach;
IRL Press: 1993.
(31) Elmore, D. T.; Smyth, J. J. Biochem. J. 1965, 94, 563-568.