Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.
gamma-Glutamylcysteine synthetase (rat kidney), which catalyzes the first step of GSH synthesis, can be dissociated into subunits (M(r) 73,000 and 27,700) by native gel electrophoresis after treatment with dithiothreitol (DTT); the heavy subunit, which exhibits catalytic activity and feedback inhibition by GSH (Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345-9347), was cloned and sequenced (Yan, N., and Meister, A. (1990) J. Biol. Chem. 265, 1588-1593). Here, the cDNA for the heavy sub unit was expressed in Escherichia coli, and the recombinant enzyme was separated from E. coli gamma-glutamylcysteine synthetase and purified. The recombinant enzyme and the isolated heavy subunit have much lower affinity for glutamate and higher sensitivity to GSH inhibition than the holoenzyme, suggesting that the heavy subunit alone would not be very active in vivo. A GSH analog, gamma-Glu-alpha-aminobutyryl-Gly (ophthalmic acid), inhibits only slightly, but inhibits much more after treatment of the holoenzyme with DTT. In contrast, ophthalmic acid inhibits the recombinant and isolated heavy subunit enzymes substantially without DTT treatment. We conclude that (a) the light subunit has a regulatory function affecting the affinity of the enzyme for glutamate and GSH and (b) feedback inhibition by GSH involves reduction of the enzyme and also competition between GSH and glutamate for the glutamate site.
Huang
C S
CS
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Chang
L S
LS
Anderson
M E
ME
Meister
A
A
D000818:Animals; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000494:pharmacology; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D046911:Macromolecular Substances; D009842:Oligopeptides; D011994:Recombinant Proteins; C016632:ophthalmic acid; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione