Kinetic Stabilities of Peptide Bonds
J. Am. Chem. Soc., Vol. 118, No. 26, 1996 6109
Table 3. Catalytic Proficiencies of Some Peptide Hydrolasesa
rate
enhancement
catalytic
t
1/2 nonenz
k
non
k
(s
cat
-1
k
(s
cat/K
M
m
-1
proficiency
(s-1)
)
-1
)
k
cat/knon
(kcat/K
)/knon (M-1)
enzyme
(years)
m
-
11
6
13
10
12
17
13
14
C-terminal peptide bond (exemplified by
AcGG) vs carboxypeptidase B, 23 °C
internal peptide bond (exemplified by AcGGNHMe)
vs angiotensin-converting enzyme, 37 °C
1100
1.8 × 10
238
6 × 10
6 × 10
1.3 × 10
1.2 × 10
1.2 × 10
3.3 × 10
b
-
9
4
19
15
1.13 × 10
13.9
1842
5.3 × 10
4.9 × 10
c
-
9
5
dipeptide bond (exemplified by GG) vs ascites tumor
1.5 × 10
7.4 × 10
d
dipeptidase, 40 °C
a
Nonenzymatic reaction rates for glycine compounds (knon) are extrapolated from the present data, to the temperature at which the enzyme
reaction was investigated. Protease reactions known to proceed through covalent intermediates, such as those catalyzed by chymotrypsin and
papain, are not included in this table, because such reactions do not permit straightforward estimation of transition state affinities from comparisons
b
c
of enzymatic and nonenzymatic reaction rates (ref 1). Carboxypeptidase B + hippuryl-L-arginine, pH 8, 23 °C (ref 18). Angiotensin-converting
enzyme + cbz-p-(NO
)Phe-His-Leu, pH 8.0, 37 °C (ref 19). d Ascites tumor dipeptidase + Ala-Gly, pH 8.3, 40 °C (ref 20).
2
significantly to the rate of hydrolysis of GG at pH 7, obtained
by extrapolation in the present work.
Peptide bonds in AcG-GNHMe, AcG-G, and G-G are
hydrolyzed at rates that differ by less than a factor of 2 at 150
angiotensin-converting enzyme) and also a dipeptidase from
ascites tumor cells are somewhat less proficient in enhancing
the rates of their respective reactions. Nevertheless, enzymes
with transition state affinities of this magnitude offer exception-
ally promising targets for the design of potent competitive
inhibitors.
The present results, summarized in Table 2, indicate that in
solvent-exposed positions in proteins, a typical C-terminal
peptide bond is hydrolyzed at a rate comparable with that of a
typical internal peptide bond. In the absence of inter- or
intramolecular catalysis, such a bond would be expected to
survive for several centuries in neutral solution at ambient
temperatures. In contrast, the peptide bond that joins an
unprotected N-terminal dipeptide to the rest of a protein chain
undergoes cleavage, to form a diketopiperazine, more than 1000-
fold more rapidly. Accordingly, a typical protein would be
expected to be completely degraded to diketopiperazines within
less time than the time required for hydrolysis of a single internal
or C-terminal peptide bond.
22
°
C, nor do their energies of activation, obtained by linear
regression analysis of the Arrhenius plots, differ by more than
the experimental error (∼1.2 kcal/mol) estimated from the
standard errors of the slopes of each of the individual plots.
Substituent effects in this series appear too insignificant to justify
any attempt at detailed interpretation. Based on an average
value of 23.8 kcal/mol for the energy of activation of all three
compounds, AcG-GNHMe, AcG-G and G-G undergo uncata-
lyzed hydrolysis at 25 °C with half-times of 600, 500, and 350
years, respectively, at pH values near neutrality.
The observed route of decomposition of GGNHMe, via rapid
formation of diketopiperazine, agrees with earlier observations
of a similar route of degradation of glycylglycine amide at 130
1
5
°
C
and of several peptides that have been shown to be
degraded by elimination of diketopiperazines from the N-
terminal position at 100 °C.16 GGNHMe cyclizes too rapidly
to permit measurement of the rate of the competing hydrolytic
cleavage of the peptide bond in GGNHMe, and GdG and
methylamine are the only products observed. In GGNHMe,
peptide hydrolysis is evidently much slower than cyclization,
so that its rate cannot be measured directly as a model for the
reaction catalyzed by an aminopeptidase. From the lack of
sensitivity of uncatalyzed peptide hydrolysis to substituent
effects, noted above, it seems reasonable to infer that hydrolysis
in GGNHMe, if it could be measured, might proceed at a rate
similar to those observed in the other cases.
With a half-time of approximately 35 days at pH 7 and 37
°C, diketopiperazine formation is rapid enough to pose an
apparent threat to the stability of proteins and to suggest a
possible rationale for the posttranslational N-acetylation of
proteins that has been observed in most proteins in higher
2
3
organisms.
It seems reasonable to speculate that in the
relatively long-lived cells of eukaryotes, N-acetylation has
evolved as a mechanism for protecting proteins against spon-
taneous degradation. Short generation times presumably al-
leviate this problem in prokaryotic organisms, in which mech-
anisms for N-acetylation appear to be absent.
Table 3 compares the present rate constants, for hydrolysis
of bonds in glycine peptides, with kinetic constants that have
been reported for proteolytic enzymes acting on peptide bonds
in similar chemical environments. By these criteria, carboxy-
peptidase B shows high proficiency as a catalyst, with (kcat/
Acknowledgment. Financial support for this work was
provided by Research Grant No. GM-18325 from the NIH.
JA954077C
(19) Hayman, S.; Patterson, E. K. J. Biol. Chem. 1971, 246, 660-669.
(20) Frick, L.; Mac Neela, J. P.; Wolfenden, R. Bioorg. Chem. 1987,
17
-1
Km)/knon ∼ 4 × 10 M . The reciprocal of this value
1
5, 100-108.
corresponds to a maximal dissociation constant of approximately
(21) The present rate constants for hydrolysis of C-N bonds in GG,
-
18
2
.5 × 10
M for the enzyme-substrate complex in the
AcGG, and AcGGNHMe in neutral solution are surprisingly similar to rate
constants reported earlier for hydrolysis of the exocyclic C-N bonds in
cytidine and adenosine.20 Bond distances (Interatomic Distances; Chemical
Society: London, 1958) suggest that exocyclic C-N bonds of cytidine and
adenosine, like peptide bonds, may be markedly stabilized by resonance.
Thus, exocyclic CN bonds in derivatives of cytosine (1.31 Å) and adenine
transition state. This Ktx value is slightly lower than those that
have been estimated for adenosine deaminase and cytidine
deaminase, which also catalyze the hydrolysis of C-N
bonds.1
7-20,21
Table 3 shows that an endopeptidase (the
(
1.30 Å) are slightly shorter than a value considered typical of the C-N
bond in peptides (1.325 Å) (Pauling, L. The Nature of the Chemical Bond;
Cornell: Ithaca, NY, 1960; pp 281-282).
(22) To reduce ambiguities of interpretation and render enzymatic and
nonenzymatic reactions more closely comparable for purposes of estimating
catalytic proficiency (see ref 1), Table 3 is limited to proteases that do not
appear act through double displacement mechanisms.
(
15) Meriwether, L.; Westheimer, F. H. J. Am. Chem. Soc. 1956, 78,
5
119-5123.
(
(
16) Steinberg, S. M.; Bada, J. L. J. Org. Chem. 1983, 48, 2295-2298.
17) Wolff, E. C.; Schirmer, E. W.; Folk, J. E. J. Biol. Chem. 1962,
2
37, 3094.
18) Stevens, R. L.; Micalizzi, E. R.; Fessler, D. C.; Pals, D. T.
Biochemistry 1972, 11, 2999-2310.
(
(23) Brown, J. L.; Roberts, W. K. 1976, 254, 1447-1454.