Chemical modification of sulfhydryl residues of rabbit triosephosphate isomerase

Rabbit muscle triosephosphate isomerase (EC 5.3.1.1) is inactivated by maleimides, Na₂S₄O₆, organic mercurials, 5,5′-dithiobis (2-nitrobenzoic acid), Ag⁺, and Hg²⁺. Ag²⁺ and Hg²⁺ cause a decrease in the maximum velocity, and under specified conditions the other reagents induce an increase in the Michaelis constant.N-ethylmaleimide reacts with three sulfhydryl residues per mole of enzyme, and the maximum change in K_m is about threefold. Mercurials cause a greater change in K_m and react with more than three sulfhydryl groups, but subsequent precipitation prevents quantitative analysis after six residues have reacted (with p-hydroxymercuribenzoate).Experiments with several competitive inhibitors and the active-site affinity label, 3-chloroacetolphosphate, showed that the magnitude of the change in Michaelis constant was the same as the magnitude of the changes in the inhibition constants.The rabbit muscle and liver enzyme appear to have similar properties, but the chicken muscle enzyme is much less reactive, and the yeast enzyme does not become inactivated.Evidence is presented to show that the effects cannot be explained by assuming the hydrated substrates are bound to the enzyme as a result of sulfhydryl modification.