Redox interconversion of glutathione reductase from Escherichia coli. A study with pure enzyme and cell-free extracts.
The glutathione reductase from E. coli was rapidly inactivated following aerobic incubation of the pure and cell-free extract enzymes with NADPH, NADH and other reductants. The inactivation of the pure enzyme depended on the time and temperature of incubation (t 1/2 = 2 min at 37 degrees C), and was proportional to the [NADPH]/[enzyme] ratio, reaching 50% in the presence of 0.3 microM NADPH and 45 microM NADH respectively, at a subunit concentration of 20 nM. Higher pyridine nucleotide concentrations were required to inactivate the enzyme from cell-free extracts. Two apparent pKa, corresponding to pH 5.8 and 7.3, were determined for the redox inactivation. The enzyme remained inactive even after eliminating the excess NADPH by gel chromatography. E. coli glutathione reductase was protected by oxidized and reduced glutathione against redox inactivation with both pure and cell-free extract enzymes. Ferricyanide and dithiothreitol protected only the pure enzyme, while NADP+ exclusively protected the cell-free extract enzyme. The inactive glutathione reductase was reactivated by treatment with oxidized and reduced glutathione, ferricyanide, and dithiothreitol in a time-and temperature-dependent process. The oxidized form of glutathione was more efficient and specific than the reduced form in the protection and reactivation of the pure enzyme. The molecular weight of the redox-inactivated E. coli glutathione reductase was similar to that of the dimeric native enzyme, ruling out aggregation as a possible cause of inactivation. A tentative model is discussed for the redox inactivation, involving the formation of an 'erroneous' disulfide bridge at the glutathione-binding site.
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005292:Ferricyanides / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology
D001426:Bacterial Proteins; D005292:Ferricyanides; D009243:NAD; C007931:hexacyanoferrate III; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol