Seok Y J, Lee B R, Zhu P P, Peterkofsky A
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):347-51. doi: 10.1073/pnas.93.1.347.
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.
糖磷酸转移酶系统（PTS）的首个蛋白质组分是64 kDa的酶I（EI），它可被磷酸烯醇丙酮酸（PEP）磷酸化，并将磷酸基团转移至受体热稳定蛋白（HPr）。大肠杆菌EI分离出的氨基末端结构域（EIN）不再被PEP磷酸化，但保留了参与向HPr进行可逆磷酸转移的能力。构建了一个表达载体用于大量生产EIN，并摸索出了使该蛋白实现最大表达的条件。本文描述了一种三步柱层析法将EIN纯化至同质；500 ml培养物可产生约80 mg纯蛋白，产率约为75%。完整的大肠杆菌EI能有效地将PEP上的磷酸基团转移至大肠杆菌的HPr，但不能转移至枯草芽孢杆菌或山羊支原体的HPr。从EI向大肠杆菌或山羊支原体的IIAglc酶（EIIAglc）进行磷酸转移需要HPr作为中间体。EIN的磷酸化形式能够进行更普遍的磷酸转移；它能将磷酸基团转移至大肠杆菌、枯草芽孢杆菌和山羊支原体的HPr，以及大肠杆菌的EIAglc。这些研究表明，EI的羧基末端结构域赋予该蛋白从PEP接受磷酸基团的能力，以及一种鉴别功能，使完整蛋白仅能促进向大肠杆菌HPr的有效磷酸基团转移。
