Malate thiokinase. Evidence for a random site reaction mechanism.

Abstract

Half-site reactivity in the malate thiokinase reaction was studied by measuring the reaction of enzyme-bound ligands in a series of single turnover experiments. A dimeric (alpha beta)2 enzyme form containing [14C] succinyl-CoA on one alpha beta subunit pair and [3H]succinyl-CoA on the adjacent alpha beta subunit pair was prepared. Reaction of this enzyme species with ATP or inorganic phosphate resulted in the release of half of the bound succinyl-CoA. The succinyl-CoA released comprised a 50-50 mixture of [14C]- and [3H]succinyl-CoA. Likewise, enzyme containing [32P]phosphate on one alpha beta subunit pair and nonradioactive phosphate on the adjacent alpha beta subunit pair reacted with ADP releasing half of the bound phosphate as a 50-50 mixture of radioactive and nonradioactive phosphate. These results serve to exclude an alternating site mechanism for the malate thiokinase reaction and support a random reaction of liganded subunits. In addition, it has been shown that enzyme containing 1 phosphate/(alpha beta)2 dimer is inactive toward phosphate transfer. However, succinyl-CoA served to activate this enzyme species for phosphate transfer. These results can be explained in terms of subunit asymmetry. The simplest model is one in which subunit asymmetry is induced upon ligand binding.