Interaction of γ-Glutamyl Transpeptidase with Amino Acids, Dipeptides, and Derivatives and Analogs of Glutathione

Abstract

Abstract A highly purified and apparently homogeneous preparation of rat kidney γ-glutamyl transpeptidase was examined with respect to its γ-glutamyl donor and acceptor specificities. Many γ-glutamyl amino acids were active as γ-glutamyl donors, the most active being l-γ-glutamyl-l-glutamine and l-γglutamyl-l-methionine. Glutathione disulfide was about 5% as active as glutathione, while substantial activity was found with S-methylglutathione and ophthalmic acid. In the course of this work convenient spectrophotometric methods for the determination of γ-glutamyl transpeptidase activity were developed in which S-substituted derivatives of glutathione were used, e.g. the S-propanone and S-acetophenone derivatives; these procedures are based on the high ultraviolet absorbance of the corresponding S-substituted products. Several of the S-substituted glutathiones were more active in transpeptidation than glutathione itself. Kinetic studies were carried out which indicate a ping-pong mechanism consistent with intermediate formation of a γglutamyl enzyme. Of the 32 amino acids tested, l-glutamine and l-methionine were the best acceptors of the γ-glutamyl group. A number of dipeptides were active as acceptors, and several of these were more active than l-glutamine, e.g. glycylglycine, glycyl-l-alanine, l-methionyl-l-serine, lglutaminyl-l-glutamine, and l-α-aminobutyrylglycine. Aminoacylglycine derivatives were (in the five instances studied) more active than the corresponding free NH2-terminal amino acids; the corresponding aminoacyl-l-alanine derivatives were less active than the aminoacylglycines. The high reactivity of certain dipeptides may reflect their affinity for the cysteinylglycine site of the enzyme, but the findings are also consistent with a function of γ-glutamyl transpeptidase in peptide transport as well as in amino acid transport. γGlutamyl transpeptidase is inhibited substantially by the γglutamyl hydrazones of a number of α-keto acids. Inhibition is competitive with respect to the γ-glutamyl donor; of seven α-keto acid γ-glutamyl hydrazones, the derivative of α-ketoglutarate inhibited the most.