Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP.
The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.
D000084:Acetate Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000345:Affinity Labels / Q000138:chemical synthesis / Q000494:pharmacology*; D002413:Cations, Divalent; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D013873:Thionucleotides / Q000138:chemical synthesis / Q000494:pharmacology*
D000345:Affinity Labels; D002413:Cations, Divalent; D010761:Phosphorus Radioisotopes; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000084:Acetate Kinase