STEADY-STATE KINETICS OF MANNITOL PHOSPHORYLATION CATALYZED BY ENZYME-II(MTL) OF THE ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM

The kinetics of mannitol phosphorylation catalyzed by enzyme II(mtl) o f 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) s olubilized and assayed above the critical micellular concentration (cm c) of the detergent, and (iii) solubilized and assayed below the cmc o f the detergent. The kinetic characteristics of enzyme II(mtl), after solubilization of cytoplasmic membranes or after purification from the se membranes are comparable. The mannitol-dependent kinetics at satura ting concentration of P-HPr were biphasic both for the solubilized enz yme assayed above the cmc and for the enzyme in ISO vesicles. In contr ast, the mannitol-dependent kinetics was monophasic for the solubilize d enzyme assayed below the cmc. In the latter case, the maximal rate w as about twice as high as observed with the two other conditions. The contribution of the high affinity phase to the maximal rate is lower f or enzyme II(mtl) 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.