Interpretation of rate profiles of the pH-dependent trypsin- and α-chymotrypsin-catalyzed hydrolysis of esters with a free α-amino group

An hypothesis is postulated to interpret the anomalous pH dependence of the rate of hydrolysis of esters with a free α-amino group catalyzed by trypsin (EC 3.4.4.4) and α-chymotrypsin (EC 3.4.4.5). This involves the supposition of a hybrid system composed of E·SαNH₃⁺ (I) + E·SαNH₂ (II), each of these having distinct constants for the three steps of the reaction. The optimum pH of System I, about 6-6.5, maybe accounted for in terms of an effect upon the enzyme of the α-ammonium group of the substrate species prevailing at low pH. It is possible that when near the enzyme this cationic group perturbs ionization equilibria of groups within the "charge relay system" of the active center. This effect appears as an acid shift of the rate profiles and apparently reinforces the catalytic activity: the rates observed in the acid region are faster in spite of high K_m values. Consequently the fall of rate around pH 7 observed in reactions of α-aminoacyl esters with trypsin and α-chymotrypsin, seems to be due to the conversion of substrate from the more reactive α-ammonium species to the less reactive α-amino species. The rate dependence for System II is apparently like that observed with "normal" substrates without a free α-amino group.