Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine synthetase

Abstract

The heavy subunit (M(r), 72,614) of rat kidney gamma-glutamylcysteine synthetase, the enzyme that catalyzes the first step of glutathione (GSH) synthesis, mediates the catalytic activity of this enzyme and its feedback inhibition by GSH. There is evidence that the light subunit has a regulatory function (Huang, C.-S., Chang, L.-S., Anderson, M.E., and Meister, A. (1993) J. Biol. Chem. 268, 19675-19680). In the present work the cDNA for the light subunit was isolated, sequenced, and expressed in Escherichia coli. The cDNA was found to code for a protein of 274 amino acid residues (M(r) 30, 548). Recombinant holoenzyme was obtained by co-expression of the heavy and light subunits and by mixing of the separately expressed proteins. These recombinant holoenzyme preparations exhibit catalytic and GSH feedback inhibitory properties that are virtually identical to those of the isolated holoenzyme. These studies establish that the light subunit is an integral part of the enzyme and that the light and heavy subunits, are coded for separately. Possibly significant similarity of sequence of amino acids was found between the light subunit and E. coli gamma-glutamylcysteine synthetase, which is a single polypeptide.