Effect of Methylamine on Trypsin Catalysis

The effect of the basic group of specific substrates of trypsin on the kinetics of the enzyme was studied. The basic group was substituted by methylamine added to a non-specific substrate. The kinetic constants of tryptic hydrolysis of N-acetyl-l-alanine ethyl ester involving methylamine, at pH 6.3–8.5 were determined. For comparison, the constants of trypsin and α-chymotrypsin were also determined. It can be concluded that methylamine increases the rate of hydrolysis catalyzed by trypsin. The relative increase of the pH-independent limiting value of (kc/Km)max is shown to be fivefold and the pKa decreases by 0.4 unit. The increase of kc is nearly fourfold. The rate constants of the hydrolysis steps catalyzed by trypsin and trypsin · methylamine, respectively, were assayed by means of the nucleophile 1,4-butanediol at pH 6.6. There is a sixfold increase of the original rates caused by methylamine for acylation (k2) and a threefold increase for deacylation (k3) but the substrate dissociation constant (Ks) was almost unchanged. A comparison of the individual rates of the methylamine-activated tryptic hydrolysis of nonspecific substrates with the appropriate data of the trypsin-catalyzed hydrolysis of specific substrates indicates that there is an approximately 1000-fold difference between the rate constants for the acylation step, while in deacylation the difference is insignificant. The methylammonium ion bound to trypsin is supposed to keep the enzyme through the catalysis in a more active conformation than the original one. This indirect effect might bear only a small part of the trypsin-substrate interaction.