Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase.
Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000494:pharmacology; D004563:Electrochemistry; D004579:Electron Transport; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives*; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000032:analysis* / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D010269:Paraquat; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry
C020857:phosphoadenosine diphosphoribose; D000246:Adenosine Diphosphate Ribose; C045716:adenosine 2',5'-diphosphate; D000244:Adenosine Diphosphate; D005980:Glutathione Reductase; D005978:Glutathione; D010269:Paraquat; D019803:Glutathione Disulfide