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, kcat(lim) for specific ester substrates increases about 50-60%, and the apparent pKa of the ionizing group on which kcat 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. Km(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 pKa 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 pKa 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, kcat(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 pKa 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, Km(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.
Áreas temáticas de ASJC Scopus
- Medicina General