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

Abstract

Enzyme I of the phosphoenolpyruvate - sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 +/- 5000 molecular weight subunits. At low protein concentration and 4 degrees C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 +/- 3 microM, 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 has been found. The amino acid composition has been determined and the epsilon 1% 280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: mechanism of the phosphoryl-group transfer from phosphoenolpyruvate to HPr

“…Treatment of EI with DNase and RNase, followed by several washing steps in an Amicon ultrafiltration apparatus with an UM-20 filter, changed the UV spectrum in that the maximum absorbance appeared at 277 nm and the minimum absorbance at 252 nm. In the modified procedure, EI eluted from the DEAE-cellulose column Whatman) a dimer molecular weight of 134000 (Misset et al, 1980;Waygood & Steeves, 1980)]. This experimental value agrees well with the extinction coefficient calculated by Waygood from the amino acid composition of enzyme I: eig$$'L = 4.4 (Waygood et al, 1980).…”

“…The mechanism of Scheme I1 differs from the one presented by Waygood & Steeves (1980). They concluded that Scheme I is the proper mechanism for the enzyme I catalyzed phosphorylation of HPr.…”

“…Since HPr and enzyme I can be purified to homogeneity (Anderson et al, 1971;Dooijewaard et al, 1979;Robillard et al, 1979;Waygood et al, 1980), detailed studies on the initial reactions in the process of phosphorylation and transport are possible. We have previously demonstrated that the active enzyme I molecuie is a dimer, which, at low concentrations, dissociates into inactivate monomers (Misset et al, 1980).…”

Understanding Glucose Transport by the Bacterial Phosphoenolpyruvate:Glycose Phosphotransferase System on the Basis of Kinetic Measurements in Vitro

“…of EI for PEP) range from 0.2 to 0.4 mM (43,65), and values of EI ⅐ P for Pyr range from 1.5 to 3 mM (66). Furthermore, the equilibrium constant for the above reaction is 1.5 (44).…”

“…First, there was a large discrepancy between reported K m values for MeGlc (170 M in vivo and 6 M in vitro) (41), whereas the K m values for glucose agreed much better (see above). Second, the K d value (Equation 43) has only been determined for glucose (42) (also see "Discussion").…”