γ Glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with [glutamate 14C]glutathione

Abstract

The hydrolytic and transfer reactions catalysed by rat kidney γ glutamyltransferase (EC 2.3.2.2) were studied in vitro with the substrates [U 14C]glutamic acid labelled glutathione and methionine. Initial velocity patterns, isotope exchange and binding studies were consistent with a branched non sequential mechanism in which a γ glutamyl enzyme intermediate may react either with water (hydrolysis) or with methionine (γ glutamyl transfer). The Michaelis constant for glutathione in hydrolysis was 13.9±1.4μM, for glutathione in transfer it was 113±15μM and for methionine as substrate it was 4.7±0.7 mM. At substrate concentrations in the ranges of their respective Michaelis constants, the rate of transfer was about ten times higher than that of hydrolysis, but at concentrations of methionine approximating to the physiological (64μM in rat plasma) the transfer is negligible. The enzyme is reported to lie on the luminal surface of the proximal straight kidney tubule. In this situation, if the kinetic results obtained with the detergent solubilized enzyme are relevant to the behaviour of the enzyme in vivo, it appears likely that the main function of renal γ glutamyltransferase is not in amino acid transport, but rather to hydrolyse glutathione in the renal filtrate.