Kinetic properties of succinylated and ethylenediamine-amidated δ-chymotrypsins

Succinic anhydride reacts with 14 of the 15 amino groups of chymotrypsinogen A, giving a derivative from which succinylated δ-chymotrypsin was prepared. Upon succinylation, k_cat(lim) for specific ester substrates increases about 50-60%, and the apparent pK_a of the ionizing group on which k_cat depends, (probably histidine 57), shifts from 7.0 in the native enzyme to 8.0 in the succinylated enzyme, as expected for the excess of negative charges in the molecule. K_m(app) values are lower than those of the native enzyme and show a very small dependence on pH which can be accounted for by the ionization of an acid group (presumably the isoleucine 16 amino group) with a pK_a of 10 in the free enzyme and 10.6 in the E-S complex. The rate constant k₃(lim) for the deacylation of the indoleacryloyl-enzyme also increases about 50% upon succinylation, and a similar shift of the histidine pK_a from 8.0 to 9.0 is observed. Direct succinylation of δ-chymotrypsin produces a rapid inactivation of the enzyme which is proportional to the disappearance of the isoleucine 16 amino group. The kinetic properties of a derivative with an excess of positive charges were also studied. Reaction of ethylenediamine with δ-chymotrypsin in the presence of a water-soluble carbodiimide resulted in the amidation of 13 carboxyl groups of the enzyme, k_cat(lim) for the hydrolysis of specific esters and k₃(lim) for the deacylation of the indoleacryloyl-amidated-enzyme were the same as those of unmodified δ-chymotrypsin. Shifts in the pK_a of the histidine upon amidation, from 7.0 to 6.1 and from 7.8 to 7.1 were observed for the hydrolysis of N-acetyl-l-tryptophan methyl ester and deacylation of indoleacryloyl-enzymes, respectively. In contrast to the native enzyme, K_m(app) values for the hydrolysis of N-acetyl-l-tryptophan methyl ester catalyzed by the ethylenediamine-amidated enzyme are strongly pH dependent. They increase sharply above pH 8.5, probably reflecting alterations in the tertiary structure of the protein as it becomes almost completely uncharged.