3-Mercaptopyruvate sulfurtransferase: rapid equilibrium-ordered mechanism with cyanide as the acceptor substrate.

A steady-state kinetic analysis of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) using cyanide as the sulfur-acceptor substrate was performed. Measurement of pyruvate production gave initial velocity patterns and secondary plots characteristic of a rapid equilibrium-ordered sequential mechanism. Initial velocity data obtained by measuring the formation of thiocyanate, which is the other reaction product, revealed a discrepancy between the rates of pyruvate and thiocyanate production; the yield of thiocyanate per unit time was smaller than that of pyruvate for each reaction mixture. This velocity discrepancy, which diminished with approach to cyanide saturation, suggests that sulfur is not discharged from the enzyme as thiocyanate, but as elemental sulfur, and that thiocyanate is formed in a subsequent nonenzymic step. A formal mechanism which has a rapid equilibrium-ordered catalytic cycle and elemental sulfur as one of the initial reaction products is proposed. Computer simulation is used to show that this model is in agreement with all of the kinetic data.