γ-Glutamyltranspeptidase-catalysed acyl-transfer to the added acceptor does not proceed via the ping-pong mechanism

Abstract

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.

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

“…1 Abbreviations: CHES, 2-[N-cyclohexylamino]ethane sulfonic acid; DON, 6-diazo-5-oxo-L-norleucine; GGT, γ-glutamyl transpeptidase (EC 2.3.2.2); GSH, glutathione (γ-Glu-Cys-Gly); kie, kinetic isotope effect; MOPS, 3-[N-morpholino]propane sulfonic acid; Tris, tris(hydroxymethyl)aminomethane. the mechanistic pathway is sequential, with random addition of substrates and ordered release of products (15). Currently, the amino acid residues involved in the enzymatic catalysis of rat kidney GGT, including the active site nucleophile, have not been identified.…”

gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405

“…The present modification experiments showed that the inactivation of the present GGT by DFP could be fully reversed by the addition of 2-ME, indicating that a cysteinyl residue and not a seryl residue was affected by DFP. The rat kidney GGT contains Thr-523, another hydroxy amino acid, which interacts with the substrate analog acivicin (8,45). Provided that more detailed studies of the present GGT support these preliminary observations, the treponemal GGT may turn out to be catalytically and structurally relatively similar to mammalian GGTs studied previously and to differ from the E. coli GGT with regard to association with a carbohydrate moiety.…”