Mechanism of Action of Guinea Pig Liver Transglutaminase: V. THE HYDROLYSIS REACTION

Abstract

Abstract The transglutaminase-catalyzed hydrolysis of p-nitrophenyl acetate is effectively inhibited, in the absence of nucleophilic amine, by another substrate for the enzyme, benzyloxycarbonyl- (Z)-l-glutaminylglycine. Kinetic evaluation of this inhibition, with consideration of the dependence on calcium ion concentration and of previous studies of the amine transfer reaction, indicates that glutamine substrate is reversibly bound to different calcium-activated forms of the enzyme in the presence and in the absence of amine. The Ki value for Z-l-glutaminylglycine as an inhibitor of the esterolysis reaction was estimated to be 1.92 ± 0.5 x 10-3 m at pH 7.0. Examination of the calcium-dependent transglutaminase-catalyzed hydrolysis of Z-l-glutaminylglycine at two pH levels showed that the mechanism of hydrolysis conforms to that suggested by the esterolysis inhibition studies. The calcium ion requirements for hydrolysis were found to be very different from those for the amine transfer reaction. The Ka values of calcium in the hydrolysis reaction, estimated as 0.6 x 10-3 m at pH 7.0 and as 2.5 x 10-3 m at pH 5.4, are significantly lower than the Ka values of calcium reported for the amine transfer reaction and the Kd values derived from calcium-induced conformational changes in the enzyme protein. Agreement of the Km values for Z-l-glutaminylglycine at two pH levels in the hydrolysis reaction (approximately 2 x 10-3 m) with the Ki value for this Z-dipeptide as an inhibitor of esterolysis is consistent with the assumption that the hydrolysis reaction proceeds by an equilibrium mechanism. The effects of glutamine substrate on the rate of reaction between iodoacetamide and an essential sulfhydryl group of calcium-activated transglutaminase were investigated. The second order rate constant for reaction of the inactivator with this essential —SH group was found to be 96 x 103 liters mole-1 min-1, either in the presence or in the absence of hydroxylamine. Studies of the calcium ion requirement for this inactivation indicated that only the form of the enzyme in which metal ion is bound at both metal-binding positions reacts with iodoacetamide under the experimental conditions used. The decrease in reactivity of the essential —SH group resulting from Z-l-glutaminylglycine binding was found to be very different in the absence and in the presence of hydroxylamine. In the absence of amine the rate was decreased to about one-half that observed without substrate, whereas in the presence of amine the essential group appeared to be protected from reaction with iodoacetamide. These manifest differences, which are a function of steric changes induced by substrate binding, are in accord with the kinetic evidence for different mechanisms of substrate binding in the hydrolysis and amine transfer reactions.