Oxidative inactivation of the enzyme rhodanese by reduced nicotinamide adenine dinucleotide.
The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is inactivated with a half-time of approximately 3 min when incubated with 50 mM NADH. NAD+, however, has virtually no effect on the activity. Inactivation can be prevented by the inclusion of the substrate thiosulfate. The concentration of thiosulfate giving half-protection is 0.038 mM. In addition, NADH, but not NAD+, is a competitive inhibitor with respect to thiosulfate in the catalyzed reaction (Ki = 8.3 mM). Fluorescence studies are consistent with a time-dependent oxidation of NADH in the presence of rhodanese. The sulfur-free form of rhodanese is more rapidly inactivated than the sulfur-containing form. Spectrophotometric titrations show that inactivation is accompanied by the loss of two free SH groups per enzyme molecule. Inactivation is prevented by the exclusion of air and the inclusion of EDTA (1 mM), and the enzyme activity can be largely protected by incubation with superoxide dismutase or catalase. Rhodanese, inactivated with NADH, can be reactivated by incubation with the substrate thiosulfate (75 mM) for 48 h or more rapidly, but only partially, by incubating with 180 mM dithiothreitol. It is concluded that, in the presence of rhodanese, NADH can be oxidized by molecular oxygen and produce intermediates of oxygen reduction, such as superoxide and/or hydrogen peroxide, that can inactivate the enzyme with consequent formation of an intraprotein disulfide. In addition, NADH, but not NAD+, can reversibly bind to the active site region in competition with thiosulfate. These data are of interest in view of x-ray studies that show structural similarities between rhodanese and nucleotide binding proteins.
D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013466:Sulfurtransferases / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D009243:NAD; D004492:Edetic Acid; D013482:Superoxide Dismutase; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase