Steady state kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.
The kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and assayed above the critical micellular concentration (cmc) of the detergent, and (iii) solubilized and assayed below the cmc of the detergent. The kinetic characteristics of enzyme IImtl, after solubilization of cytoplasmic membranes or after purification from these membranes are comparable. The mannitol-dependent kinetics at saturating concentration of P-HPr were biphasic both for the solubilized enzyme assayed above the cmc and for the enzyme in ISO vesicles. In contrast, the mannitol-dependent kinetics was monophasic for the solubilized enzyme assayed below the cmc. In the latter case, the maximal rate was about twice as high as observed with the two other conditions. The contribution of the high affinity phase to the maximal rate is lower for enzyme IImtl in ISO vesicles than for the solubilized enzyme. At limiting concentrations of P-HPr, the kinetics is not according to the expected "ping-pong" mechanism.
Lolkema
J S
JS
Department of Biochemistry, University of Groningen, The Netherlands.
ten Hoeve-Duurkens
R H
RH
Robillard
G T
GT
D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D014316:Tritium
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D014316:Tritium; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli