A STUDY OF THE STABILIZATION OF TETRAHEDRAL ADDUCTS BY TRYPSIN AND DELTA-CHYMOTRYPSIN

Delta-chymotrypsin has been alkylated by 1-C-13- and 2-C-13-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-C-13- and 2-C-13-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pK(a) values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-C-13- and 2-C-13-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-C-13-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pK(a) of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the C-13 titration shifts of these model compounds with those of the C-13 enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that. in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10000-fold greater than it would be in water. We conclude that the pK(a) of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pK(a) of the imidazolium cation of histidine-57 is > 7.9 and > 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pK(a) of the imidazolium cation is > 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pK(a) of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195. A molecular model and kinetic scheme is presented which explains why this increase in the pK(a) values of the imidazolium cation is not manifested in the pK(a) values determined from the pH dependence of k(cat.) values and why these pK(a) values are generally less than pK(a) values determined from the pH dependence of k(cat.)/K(m) values.