Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkylmaleimides.
A series of straight chain N-alkymaleimides was shown to simultaneously inactivate the reductase, transhydrogenase and diaphorase activities of yeast glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2.) at pH 7.5 and 25 degrees C. Apparent second-order rate constants for the inactivation of all enzyme activities exhibited parallel increases with increasing chainlength from C-2 through C-7 of the alkyl substituent of the enhanced binding of maleimides through nonpolar interactions with the enzyme. Reduction of the active site disulfide with NADPH was required prior to addition of maleimide for inactivation to occur. NADP, AcPyADP, SNADP, AADP, and oxidized glutathione all protected the enzyme from inactivation. 2'AMP, 3' AMP, 2'-phospho-5' AMP, 2'-phospho5'-ADP and 2'-phospho-ADP-ribose although all coenzyme-competitive inhibitors failed to protect the enzyme from N-ethylmaleimide inactivation. N-Phenyl and N-alkylmaleimides covalently modified two, of six available sulfhydryl groups per subunit. No other amino acid residues were modified. The reactivity of these sulfhydryl groups was at least two orders of magnitude higher than any reported for the N-ethylmaleimide reaction with many other 'essential sulfhydryl' enzymes. No change in the charge transfer band of the reduced enzyme was observed upon complete inactivation by N-ethyl, N-heptyl or N-phenylmaleimide. The retention of the charge transfer band after selective modification of two sulfhydryl groups suggests the involvement of a third reactive sulfhydryl group in the functioning of the yeast enzyme. No inactivation was observed when coenzymatically reduced enzyme was incubated with the site-specific sulfhydryl reagent, diazotized AADP.
D000478:Alkylation; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005292:Ferricyanides / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors*; D008301:Maleimides / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009941:Organomercury Compounds / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D005292:Ferricyanides; D005452:Fluoresceins; D008301:Maleimides; D009941:Organomercury Compounds; D009243:NAD; C001673:fluorescein mercuric acetate; D009249:NADP; C024375:3-aminopyridine adenine dinucleotide phosphate; D004228:Dithionitrobenzoic Acid; D005980:Glutathione Reductase