JBC, Vol. 250, Issue 14, 5393-5399, Jul, 1975
Modification of isoleucine-16 acetylated delta-chymotrypsin
S. Kumar, K. Dar, S. Ganno and H. Hatano
Activation of acetylated chymotrypsinogen with trypsin leads to
catalytically active acetylated delta-chymotrypsin containing NH2-terminal
isoleucine. The importance of the cationic terminus to the control of the
active conformation of acetylated delta-chymotrypsin has been demonstrated
(Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem.
241, 2720). Later studies appeared to suggest that the modification of
isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of
catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine
ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A.
(1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A.,
Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss
of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970)
Biochemistry 9, 3902). In the present studies, controlled acetylation of
the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic
anhydride led to a progressive loss of active sites of the enzyme.
Determination of the catalytic and kinetic properties of the modified
enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or
the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole
gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as
substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus
or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are
nearly identical and the kcat value is reduced to about 25% in the latter
enzyme species. This value correlates well with about 20% of the active
sites in this enzyme as measured by the rapid initial liberation of
p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate
constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3%
acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25
degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16
and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity
could be correlated well with the residual NH2-terminal isoleucine content
and with the moles of [1--14C]acetyl groups incorporated per mol of the
enzyme. The activity associated with the modified enzyme can be attributed
to the enzyme species in which isoleucine-16 of acetylated
delta-chymotrypsin is not acetylated. These data are in general agreement
with the studies of Ghelis et al. (1970) but are in disagreement with the
results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the
hypothesis that the final conformation of acetylated delta-chymotrypsin
containing an acetylated NH2 terminus is catalytically inactive and
resembles acetylated zymogen in many of its physical properties.
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