Reaction of malate thiokinase with methoxycarbonyl-CoA disulfide. Evidence for half-of-the-sites reactivity.
Reaction of the active site-directed sulfhydryl reagent methoxycarbonyl-CoA disulfide with malate thiokinase follows pseudo-first order kinetics and leads to a complete loss in enzyme activity as measured by the overall reaction. The rate of inactivation of enzyme in buffer alone shows a linear dependence on the methoxycarbonyl-CoA disulfide concentration. In the presence of the substrate phosphate or the substrate analog sulfate, the rate of enzyme inactivation displays a hyperbolic response with respect to methoxycarbonyl-CoA disulfide. Anions such as chloride and fluoride decrease the rate of enzyme inactivation. Low concentrations of succinyl-CoA protect the enzyme against inactivation, while L-malate protection of the enzyme is observed only in the absence of added sulfate. Protection of the enzyme by ATP can be attributed to phosphorylation of the enzyme and a decrease in reactivity of the phosphoenzyme relative to the native enzyme. Enzyme reacted with excess methoxycarbonyl-CoA disulfide, although inactive with respect to turnover, catalyzes ATP-ADP exchange at one-half the rate of the native enzyme. The inactive enzyme can be phosphorylated by ATP, but only to 50% the extent of native enzyme. Titration of the enzyme with methoxycarbonyl-CoA disulfide shows that the enzyme exhibits all-of-the-sites reactivity with respect to this reagent. A correlation of titration of the enzyme with methoxycarbonyl-CoA disulfide with the loss in phosphorylation site shows that reaction of methoxycarbonyl-CoA disulfide produces half-of-the-sites reactivity with respect to enzyme phosphorylation.
D001665:Binding Sites; D003065:Coenzyme A / Q000031:analogs & derivatives* / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D012492:Salts
D046911:Macromolecular Substances; D012492:Salts; C022029:methoxycarbonyl-coenzyme A disulfide; D003066:Coenzyme A Ligases; C021537:malate - CoA ligase; D003065:Coenzyme A