Escherichia coli acetate kinase mechanism studied by net initial rate, equilibrium, and independent isotopic exchange kinetics.

The equilibrium constant of the reaction catalyzed by acetate kinase (E.C. 2.7.2.1.) was determined: K = (MgADP) (acetylphosphate)/(MgATP)(acetate) = 6.7 +/- 1.3 X 10 (-4) (pH 7.4, 25 degrees). The respective free nucleotides uncomplexed to magnesium inhibit the net reaction in both directions: competitively with the respective magnesium nucleotide (MgATP or MgADP) and noncompetitively with the co-substrate (acetate or acetylphosphate). Excess free magnesium also inhibits the net reaction in both directions. The inhibition is not competitive with the phosphoryl donor (MgATP or acetylphosphate) but is competitive with respect to the phosphoryl acceptor (acetate or MgADP). A 50-fold increase in concentration of reactants at equilibrium in 0.1 M Tris/HCl, pH 7.4, at 25 degrees resulted in a rise to plateau levels of the acetate equilibrium acetylphosphate exchange rate (measured with [U-14C]acetate) and of the ATP equilibrium ADP exchange rate (measured with [U-14C]ADP and 10-fold higher than acetate equilibrium acetylphosphate), suggesting that there is no compulsory order of binding of magnesium nucleotide and co-substrate and that all chemical transformation steps cannot be much slower than the dissociation steps. The ATP equilibrium ADP exchange rate was independent of the presence or concentration of co-substrate, whereas the acetate equilibrium acetylphosphate exchange reaction occurred only in the presence of magnesium nucleotide, and the rate was directly related to the degree of saturation of enzyme with magnesium nucleotide. The independent ATP equilibrium ADP exchange, which presumably involves a phosphoenzyme intermediate, was progressively inhibited by increasingly elevated MgADP concentrations (when MgADP/MgATP greater than or equal 5), but increasing MgATP/MgADP was not inhibitory, suggesting that MgADP may bind to nonphosphorylated as well as phosphorylated enzyme, but that MgATP cannot bind to phosphorylated enzyme. While direct transfer of the phosphoryl group between nucleotide and co-substrate in a concerted mechanism has not been ruled out, an "activated ping pong" mechanism can also be proposed which is compatible with the isotopic exchange and initial net rate kinetic results. This mechanism includes a phosphoenzyme intermediate and requires enzyme-bount MgADP for phosphorylation of the enzyme by acetylphosphate.