Half-of-the-sites reactivity in the malate thiokinase reaction.

Malate thiokinase catalyzes the reversible formation of malyl-CoA or succinyl-CoA from ATP, CoA, and malate or succinate, respectively. Incubation of the enzyme with succinyl-CoA results in the formation of a tight complex in which 4 succinyl-CoA molecules are bound/alpha 4 beta 4 tetramer. Denaturation of the enzyme with urea or sodium dodecyl sulfate releases succinyl-CoA from the enzyme. Enzyme-bound succinyl-CoA is also released by reaction with ADP + Pi (reverse reaction) or by reaction with arsenate (arsenolysis reaction). Addition of ATP to enzyme containing 4 mol of bound succinyl-CoA/mol of enzyme leads to the loss of 2 of the molecules of bound succinyl-CoA with the concomitant incorporation of 2 molecules of phosphate into the enzyme. In a single turnover experiment in which enzyme containing 2 mol of bound succinyl-CoA and 2 mol of phosphate/mol of alpha 4 beta 4 tetramer was incubated with succinate plus CoA, only phosphate was released from the enzyme. Isolation of the enzyme from a reaction mixture undergoing catalysis showed 2 mol of succinyl-CoA bound/enzyme tetramer. These results suggest that, during steady state catalysis, approximately 2 molecules of succinyl-CoA are bound/tetramer, and that malate thiokinase exhibits half-of-the-sites reactivity.