Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate.
Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S. R. (1957) Biochim. Biophys. Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits. Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role. PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability. Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction. At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate. The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate.
Ahn
K
K
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307.
Kornberg
A
A
D001345:Autoradiography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor)
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase