Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties

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Abstract
Enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) was purified to homogeneity from E. coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a 2-fold increase in the amount of activity was used. The enzyme is a dimer of 67,000 .+-. 5000 MW subunits. At low protein concentration and 4.degree. C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2-10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for [protein] HPr of 9 .+-. 3 .mu.M, a Km for phosphoenolpyruvate of 0.18 .+-. 0.04 mM and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity was found. The amino acid composition was determined and the .epsilon.1%280 nm [specific extraction coefficient at 280 nm] is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.