JBC, Vol. 252, Issue 5, 1633-1638, Mar, 1977
Mechanisms of acylation of chymotrypsin by phenyl esters of benzoic acid and acetic acid
C. D. Hubbard and T. S. Shoupe
The kinetics of the acylation of alpha-chymotrypsin by a series of
substituted phenyl p-nitrobenzoates have been studied by stopped flow and
conventional spectrophotometry. Electron withdrawal in the leaving group
accelerates the rate of acylation, and the p value obtained for eight
esters is +1.96. The pH- and pD-independent acylation rate constants are,
respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl
p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3)
M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of
structure-reactivity results and kinetic solvent isotope effects indicates
a mechanism for acylation by phenylbenzoates in which initial reaction is a
nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently,
there is rapid transfer of the acylating group to the serine 195 from the
acylimidazole species. The kinetic solvent isotope effects for acylation by
p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and
p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0,
respectively. The latter ester is inhibited more than is p-nitrophenyl
benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as
co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic
solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and
p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate
ester is unaffected. The results for the latter substrate are in accord
with a general base-catalysed mechanism. Electron-withdrawal groups in the
phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a
leaving group p of 2.05. The kinetic solvent isotope effects for acylation
by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to
2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not
unambiguously defined using these data.
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