Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.