Y-Glutamyltranspeptidase-Catalysed Acyl-Transfer to the Added Acceptor Does Not Proceed via the Ping-Pong Mechanism

Acyl-transfer catalysed by gamma-glutamyltranspeptidase from bovine kidney was studied using gamma-L- and gamma-D-Glu-p-nitroanilide as the donor and GlyGly as the acceptor. The transfer of the gamma-Glu group to GlyGly was shown to be accompanied by transfer of the gamma-Glu group to water (hydrolysis). The results were compared with acyl-transfer catalysed by the representative serine protease, alpha-chymotrypsin. The main difference between the kinetic mechanism of the acyl-transfer reactions catalysed by these enzymes, which contain an active-site serine and form an acyl-enzyme intermediate but belong to different enzyme classes, was found to consist in the role of the enzyme-donor-acceptor complex. This complex is not formed at any acceptor concentrations in the acyl-transfer reactions catalysed by the serine proteases. In contrast, in the gamma-glutamyltranspeptidase-catalysed acyl-transfer the pathway going through the ternary enzyme-donor-acceptor complex formed from the enzyme-acceptor complex becomes the main pathway of the transfer reaction even at moderate acceptor concentrations. As a result, gamma-glutamyltranspeptidase catalysis follows a sequential mechanism with random equilibrium addition of the substrates and ordered release of the products. The second distinction concerns the inhibitory effect of the acceptor. In the case of alpha-chymotrypsin this was the result of true inhibition, i.e. a dead-end formation of the enzyme-acceptor complex. A salt effect caused by the acceptor was the rationale of a similar effect observed in acyl-transfer catalysed by gamma-glutamyltranspeptidase

Steady State Kinetics of Glutamine Cyclotransferase

The reaction mechanism of papaya latex glutamine cyclotransferase was studied using pH and temperature dependencies, a proton inventory technique, and molecular modeling. The pH-dependence of the Michaelis-Menten parameters showed that the published pH dependence of the enzyme activity was mainly the result of pH-dependent change of the active (unprotonated) substrate concentration. The enzyme activity as such changed very slightly in the pH range between 4.5 and 10. The solvent kinetic isotope effect reflected a change in Vm while the proton inventory was found to be linear with the fractionation factor of the exchangeable proton in the transition state of 0.785. The results were not consistent with an acyl-enzyme mechanism, but rather favored a simple intramolecular cyclization of the glutamine residue to the pyroglutamic acid residue. The mechanism proposed consists of the following main steps: (i) intramolecular nucleophilic attack on the γ-C=O carbon by the nitrogen of the α-amino group, (ii) transfer of a proton from the α-amino group to the nitrogen of the amide group, facilitated by an acidic group of the enzyme, and (iii) expulsion of the ammonia-leaving group promoted by this or another acidic enzyme group