Affinity labeling of gamma-glutamyl transpeptidase and location of the gamma-glutamyl binding site on the light subunit.

Abstract

.gamma.-Glutamyl transpeptidase, which consists of 2 nonidentical subunits, is rapidly inactivated with respect to its transpeptidase and hydrolase activities by the .gamma.-glutamyl analogs 6-diazo-5-oxo-L-norleucine and L-azaserine. Inactivation, which is prevented by .gamma.-glutamyl substrates (but not by acceptor substrates), is accelerated by maleate, which was previously shown to enhance utilization of glutamine by transpeptidase. 6-Diazo-5-oxo-norleucine reacts specifically, covalently and stoichiometrically at the .gamma.-glutamyl site of the enzyme, which was localized through studies with 6-diazo-5-oxo-[14C]norleucine, to the light subunits of both the transpeptidase of rat kidney (which has subunits of MW 22,000 and 46,000) and the transpeptidase of human kidney (which has subunits of MW 22,000 and 62,000). The findings, which indicate that these enzymes have similar .gamma.-glutamyl binding subunits, are relevant to the structure-function relationships of this membrane-bound enzyme and its physiological role.