Latent active site in rat-kidney gamma-glutamyl transpeptidase. The refolding process of the large subunit and characterization of the renatured enzyme.

Abstract

Rat kidney γ-glutamyl transpeptidase (EC 2.3.2.2) is composed of two non-identical subunits (the large and small subunits). In the native state of this enzyme, the active site resides on the small subunits. The present investigation has been performed to confirm and extend in further detail the previously described finding that the large subunit isolated from the native dimeric enzyme is also catalytically active [S. Horiuchi, M. Inoue and Y. Morino (1978) Biochem. Biophys. Res. Commun. 80, 873-878]. A pure preparation ofγ-glutamyl transpeptidase which had been completely inactivated by affinity labeling with 6-diazo-5-oxo-l-norleucine was dissociated into the small subunit and the large subunit under denaturating conditions. Upon renaturation, only the large subunit restored the enzymic activity. Sephadex G-200 column chromatography of the renatured preparation of the large subunit revealed the presence both of enzymically inactive aggregate forms and the enzymically active species. The enzymically active molecules of the renatured large subunit behaved as particles having a molecular weight of 48000 ± 2000. This indicates that the enzymically active species of the large subunit has a monomeric structure. Under the present experimental conditions, the restoration of enzymic activity of the large subunit was optimal at a protein concentration of approximately 150 μpg/ml and seemed to require the presence of glutathione or its S-methyl derivative as a substrate ligand. The restoration of the enzymic activity of the large subunit was accompanied by a conformational change as judged by both circular dichroic and immunochemical properties. Some kinetic properties of the reaction with γ-glutamyl-p-nitroanilide and the affinity label, 6-diazo-5-oxo-l-norleucine were essentially identical between the renatured large subunit and the native oligomeric enzyme.