8910
J. Am. Chem. Soc. 1998, 120, 8910-8913
The pH-Rate Profile for the Hydrolysis of a Peptide Bond
Robert M. Smith and David E. Hansen*,‡
†
Contribution from the Molecular and Cellular Biology Program, UniVersity of Massachusetts, Amherst,
Massachusetts 01003, and Department of Chemistry, Amherst College, Amherst, Massachusetts 01002
ReceiVed February 10, 1998. ReVised Manuscript ReceiVed June 29, 1998
Abstract: The rate of hydrolysis of N-(phenylacetyl)glycyl-D-valine (PAGV), an acyclic penicillin G analogue,
at pH 0, 1, 3, 5, 7, 9, 11, 13, and 14 has been measured at 37 °C and a pH-rate profile constructed. At each
pH, hydrolysis of both the (phenylacetyl)glycyl amide bond and glycyl-D-valine peptide bond was monitored.
At pH 3, 5, 7, 9, and 11, the hydrolysis products glycyl-D-valine and D-valine were derivatized with naphthalene-
2,3-dialdehyde in the presence of cyanide; the resultant 1-cyano-2-substituted-benz[f]isoindole (CBI) derivatives,
which are highly fluorescent, were then quantified using reverse-phase HPLC. The hydrolysis reactions were
explicitly shown to be first-order in peptide concentration at pH 5 and 9, and all rates were shown to be
independent of the buffer concentration. The rates at pH 0, 1, 13, and 14 were measured in 1 M DCl, 0.1 M
1
DCl, 0.1 M NaOD, and 1 M NaOD, respectively, and the hydrolysis products were detected by H NMR. The
first-order rate constants obtained from the above reactions were fit to the general equation k ) kH 0 +
2
+
+
-
-
kH 0 [H3O ] + kOH [OH ] to yield the following results: for hydrolysis of the (phenylacetyl)glycyl bond,
3
-
11 -1
+
-6
-1 -1
-
-6
kH O ) (9.05 ( 6.36) × 10
s , kH O ) (1.60 ( 1.04) × 10
M
s , and kOH ) (1.11 ( 0.73) × 10
-11 -1
+
3
2
-
3
1
-1
M
0
s ; and for hydrolysis of the glycyl-D-valine bond, kH O ) (8.23 ( 4.33) × 10
s , kH O ) (1.67 (
2
-
6
-1 -1
-
-6
-1 -1
.80) × 10
M
s , and kOH ) (1.16 ( 0.56) × 10
M
s . At pH 7, the hydrolysis of both the
(
phenylacetyl)glycyl amide bond and glycyl-D-valine peptide bond is dominated by kH O. The corresponding
2
half-life for (phenylacetyl)glycyl bond hydrolysis is 243 years (with a range of 143-817 years within
experimental error), while that for glycyl-D-valine bond hydrolysis is 267 years (with a range of 175-564
years).
5
Introduction
In 1988, Kahne and Still published the pH-rate profile for
14
the hydrolysis of the tetrapeptide (phenylalanyl)3[ C]glycine,
linked via its amino terminus to a polyacrylamide resin; the
release of radiolabeled products from the solid support was
monitored. Only the carboxy-terminal phenylalanylglycine
peptide bond was observed to undergo hydrolysis, and the
absence of endo-peptide bond cleavage was attributed either to
the conformational flexibility of glycine (and, thus, the increased
likelihood of intramolecular anhydride formation) or to an
artifact of the solid-phase system. Over the pH range 4-10,
the rate of hydrolysis was found to vary by less than 1 order of
Except at extremes of pH and temperature, nonenzymatic
peptide bond hydrolysis is a slow process that is difficult to
quantify accurately. Recently, however, we described a ho-
mogeneous assay for the direct measurement of this rate under
1
mild conditions. (This work was stimulated by our interest in
identifying antibodies with sequence-specific peptidase activ-
2
ity. ) In the assay, which is based upon that of Stobaugh and
co-workers,3 the newly generated R-amino group of each
hydrolysis product is derivatized with naphthalene-2,3-dialde-
hyde in the presence of cyanide ion (NDA/CN).4 The resultant
+
magnitude; a first-order dependence on [H ] was observed at
-
1-cyano-2-substituted-benz[f]isoindole (CBI) derivatives, which
pH < 3, and on [OH ] at pH > 10. At pH 7 and 25 °C, the oft
-
9
-1
are highly fluorescent, are then quantified using reverse-phase
cited rate constant of 3 × 10
s
was determined, which
HPLC. We now report the use of NDA/CN assay (at pH 3, 5,
corresponds to a half-life of approximately 7 years. That this
value may be artificially high was noted by Kahne and Still,
but a series of elegant control experiments suggested that the
rate constant measured does indeed correspond to hydrolysis
of the endo-phenylalanylglycine peptide bond.
1
7
, 9, and 11), as well as H NMR spectroscopy (at pH 0, 1, 13,
and 14), to construct the pH-rate profile for the hydrolysis of
N-(phenylacetyl)glycyl-D-valine, an acyclic analogue of penicil-
lin G.
In 1996, two additional reports on peptide bond hydrolysis
appeared. Using the NDA/CN assay, our group measured a
†
1
Molecular and Cellular Biology Program.
Department of Chemistry.
‡
-10 -1
rate constant of 1.3 × 10
s
at pH 9 and 25 °C for the
(
1) Bryant, R. A. R.; Hansen, D. E. J. Am. Chem. Soc. 1996, 118, 5498-
499.
2) (a) Smith, R. M.; Weiner, D. P.; Chaturvedi, N. C.; Thimblin, M.
D., Jr.; Raymond, S. J.; Hansen, D. E. Bioorg. Chem. 1995, 23, 397-414.
b) Yuan, P.; Plourde, R.; Shoemaker, M. R., Moore, C. L.; Hansen, D. E.
J. Org. Chem. 1995, 60, 5360-5364.
3) (a) de Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.;
Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096-
101. (b) de Montigny, P.; Riley, C. M.; Sternson, L. A.; Stobaugh, J. F.
J. Pharm. Biomed. Anal. 1990, 8, 419-429.
4) Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; Matuszewski, B.
K. J. Org. Chem. 1986, 51, 3978-3983.
hydrolysis of the peptide bond in hippurylphenylalanine, a
carboxypeptidase A substrate. Radzicka and Wolfenden6
studied the hydrolysis of acetylglycylglycine N-methylamide,
acetylglycylglycine, and glycylglycine at temperatures ranging
from 100 to 180 °C. Rate constants at pH 6.8 and 25 °C of 3.6
5
(
(
(
-11
-1
-11
-1
-11
-1
×
10
s
, 4.4 × 10
s
, and 6.3 × 10
s ,
1
(5) Kahne, D.; Still, W. C. J. Am. Chem. Soc. 1988, 110, 7529-7534.
(6) Radzicka, A.; Wolfenden, R. J. Am. Chem. Soc. 1996, 118, 6105-
(
6109.
S0002-7863(98)00456-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/21/1998