Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides. The coupling between transport and phosphorylation in inside-out vesicles

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Abstract
The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has been questioned. The coupling was tested here by analyzing the kinetics of fructose phosphorylation by inside-out vesicles of R. sphaeroides. It was concluded that fructose has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The Km of the phosphorylation reaction is 8 .mu.M. The diffusion of fructose into the vesicle is also catalyzed by the components of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a Km of 4 mM and is rate-limiting over a large external fructose concentration range. In summary, there is no phosphorylation without transport, but there is transport without phosphorylation.