Essential arginine residues in the pyridine nucleotide binding sites of glutathione reductase.
Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) from human erythrocytes as well as from other sources was inactivated irreversibly by the arginine modifying reagents 2,3-butanedione, 1,2-cyclohexanedione and phenylglyoxal. The inactivation caused by these reagents increased with time and was biphasic in nature. Inactivation by 2,3-butanedione in borate buffer could be partially reversed by dialysis against phosphate buffer or by incubation with hydroxylamine. The dicarbonyl reagents simultaneously inactivated the transhydrogenase activity of glutathione reductase that is expressed by the pyridine nucleotide sites. Amino acid analysis of enzyme modified with 2,3-butanedione showed a decrease by about 5 in the number of arginine residues. Analysis of enzyme inactivated with phenylglyoxal indicated modification of two arginine residues. NADPH, NADP/ and other adenosine 2'-phosphate nucleotides partially protected the enzyme against inactivation. GSSG did not protect the enzyme significantly. Modified glutathione reductase displayed a diminished affinity for the nucleotides as shown by affinity chromatography on 2',5'-ADP-Sepharose and by stopped-flow kinetics of the reduction of the enzyme. Modification did not cause any gross structural changes of enzyme molecule. It is concluded that some of the arginine residues modified are located at the pyridine-nucleotide-binding sites of the enzyme.
D000227:Adenine Nucleotides / Q000494:pharmacology; D001120:Arginine; D001665:Binding Sites / Q000187:drug effects; D003931:Diacetyl / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000097:blood*; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism* / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D013057:Spectrum Analysis
D000227:Adenine Nucleotides; D009249:NADP; D001120:Arginine; D005980:Glutathione Reductase; D005978:Glutathione; D003931:Diacetyl; D010658:Phenylglyoxal; D019803:Glutathione Disulfide