Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.
The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.
Seok
Y J
YJ
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Lee
B R
BR
Zhu
P P
PP
Peterkofsky
A
A
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000201:enzymology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase