The differential functional stability of various forms of bovine liver rhodanese.
Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.
Aird
B A
BA
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Horowitz
P M
PM
D000818:Animals; D000838:Anions / Q000494:pharmacology; D002417:Cattle; D008099:Liver / Q000201:enzymology*; D011190:Potassium Cyanide / Q000494:pharmacology; D012996:Solutions; D013466:Sulfurtransferases / Q000502:physiology*; D013884:Thiosulfate Sulfurtransferase / Q000502:physiology*; D013885:Thiosulfates / Q000494:pharmacology; D013997:Time Factors
D000838:Anions; D012996:Solutions; D013885:Thiosulfates; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C017717:sodium thiosulfate; D011190:Potassium Cyanide