Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at least one experimentally established phosphoenzyme (E-P) intermediate on the reaction pathway. Sterically, the phosphoryl transfer occurs with a net inversion of the configuration of the phosphorus atom. These facts are best in accord with a triple-displacement mode of action for acetate kinase, with two E-P intermediates and three steric inversions on phosphorus. It follows that a second E-P for acetate kinase must exist.Ever since its discovery by Lipmann (1), acetate kinase (EC 2.7.2.1) has engaged the active interest of students of enzyme mechanism, and never more so than now. The enzyme catalyzes the synthesis of ATP from acetyl-P. In the reverse direction the enzyme is the prototypic carboxylate kinase. As such, the chemical means by which acetate kinase effects phosphoryl transfer has been much studied, with broad implications in the wider field of phosphotransferases generally.REVIEW OF AVAILABLE DATA The equilibrium constant of reaction 1, as catalyzed by the Escherichia coli enzyme, is reported to range from 200 to 1500 (1-5), indicating that, thermodynamically, the reaction greatly favors ATP synthesis from acetyl-P. In its specificity, the enzyme is most active on acetyl-P but has some activity on propionyl-P (30%) and carbamoyl-P (18%), and none on butyryl-P, glutaryl-P, and phosphoenolpyruvate (4). The purines GTP, ITP, and dATP, as well as ATP, are excellent substrates (2, 4), accounting thus for the intrinsic purine nucleoside-5'-diphosphate kinase (NDP kinase, EC 2.7.4.6) activity of acetate kinase (6, 7).It is curious that the Km for acetate is 300 mM (2), one of the highest Michaelis constants on record. Another curiosity is the cold inactivation to which acetate kinase is liable (8). Chilled at 0°C, the enzyme quickly loses activity. Restoration of activity is possible; it is most rapidly (2-3 min) achieved, even at 0°C, by the addition of nucleotide (4). Acetate and acetyl-P are without effect. Reactivation in the absence of nucleotide requires a long-term (1-2 hr) incubation at room temperature in the presence of Mg2+ and a monovalent cation (7,8). If these inactivation-reactivation phenomena are due to conformational changes in the protein, as seems probable, then it is clear that only the nucleotides, of the four substrates of reaction 1, can specifically induce the active conformation of the enzyme.Phosphorylation of Acetate Kinase. Fig. 1 shows that the enzyme can be phosphorylated with ATP or acetyl-P, and the phosphoenzyme (E-P) can be isolated by gel filtration (6, 9, 10). The E-P prepared with ATP and the E-P prepared with acetyl-P are identical with respect to pH-stability, sensitivity to hydroxylamine, and the capacity to phosphorylate ADP or acetate (4). The phosphoryl group in E-P is linked to a carboxyl of the enzyme (9). Reduction of E-P with boro[3H]hydride (and isolation, after hydrolysis, of tritiated a...
