Active site phosphorylation of enzyme I of the bacterial phosphotransferase system by an ATP-dependent kinase.
Enzyme I (EI) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is autocatalytically phosphorylated by P-enolpyruvate. We report here an ATP-dependent kinase (EI-K) from Escherichia coli that reversibly phosphorylates EI at its active site histidine; ATP and EI-K can therefore replace P-enolpyruvate. EI-K contains a bound cofactor that is lost during purification with concomitant loss of activity. NAD+ and NADP+ substitute for the cofactor and restore activity to the apoenzyme, whereas their analogues are inactive. The pyridine nucleotides do not activate EI-K by covalent modification (e.g. ADP-ribosylation), but must be present during the kinase reaction. NADH and NADPH are potent inhibitors of EI-K at all stages of purity, and enzyme activity in a mixture of NAD+ and NADH depends on the ratio of the two pyridine nucleotides. Inhibition is observed with reduced beta-NMN and alpha-NADH, but neither is as effective as beta-NADH. The reverse reaction, the transfer of the phosphoryl moiety from phospho-EI to ADP, also requires NAD+ or NADP+. In the absence of NAD+ or NADH, [32P]phospho-EI is hydrolyzed to 32Pi, suggesting that EI-K can act as a phospho-EI phosphatase. EI kinase may serve as a link between PTS-driven sugar transport and the electron transport chain.
Dannelly
H K
HK
Department of Biology and the McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Roseman
S
S
D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006639:Histidine; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology
D009243:NAD; D006639:Histidine; D009249:NADP; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011188:Potassium