The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides

Abstract

Two interrelated sites have been detected on the fructose carrier in Rhodopseudomonas sphaeroides: an activitylinked dithiol and a Zn2+-binding site. Binding of Zn2 + brings ET; ' into a new conformation that to some extent mimics the conformation of phosphorylated Ef;", an essential intermediate in the turnover of the enzyme. Binding of zinc to Ef;'" or phosphorylating the enzyme protects it against trypsin inactivation relative to thc dephosphorylated zinc-free enzyme.A dithiol is essential for activity. lnterchanges between the redox states of the enzyme can be brought about by dithiothreitol and ferricyanide, but not, or very slowly, by molecular oxygen. The dithiol is protected, in the ECU-Zn2+ complex, against (a) alkylation by MalNEt, (b) reversible oxidation by Fe(CN):-and C u 2 + , (c) irreversible oxidation by Cu2+. The pKvalue of the activity linked thiol is 7.8. Protection experiments show that the dithiol is not located in any of the substrate-binding sites. The redox state of the enzyme does not influence the rate of inactivation of E;' " by trypsin.Fructose is transported into the bacterium Rhodopseudo-M ?~I Z C I S sphaeroides by a phosphoepzolpyruvate-dependent phosphotransferase system. The overall reaction Fru,,, + P-enolpyruvate + Fru-1-Pi, + pyruvate is catalysed by two protein species: an integral membrane protein, Ef;'", and a component that is complexed to it, called SF (soluble factor). Both are dimers with molecular mass of 110 kDa and 220 kDa respectively. J I~ virro the two components exist in an association/dissociation equilibrium that is slow compared to the turnover of the system. The complex can, therefore, be regarded as the catalytically active species. The mechanism of the phosphoryl group transfer from P-enolpyruvate to fructose has been shown to proceed via phosphorylation of the two proteins [l].The transport of a substrate over the membrane of a cell is the very first step in the metabolism of that substrate. Transport systems are driven by various energy sources, dpH, dip, ATP, P-erzolpyruvatc and solute concentration gradients. The different energy-coupling mechanisms have introduced differences in the systems on a molecular level; however, it is quite possible that the actual translocation of the substrate through the carrier protein follows the same mechanism in these various systems. We think that the phosphotransferase systems provide a good opportunity for the elucidation of this mechanism for two reasons. The first is that the measurement of the activity of the carrier, Ell, does not require compartmentalization. Phosphorylation of fructose is strictly coupled to the transport offructose [2] for the fructose-specific system in Rps. sphaeuoidcs. Therefore, phosphorylation activity also measures transport activity, even though there are no separale compartments. The second reason is that, during turnover, the carrier protein cycles through two well-defined states: the phosphorylated and unphosphorylated state.Phosphorylation of E,* causes a conformational change in t...

Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

“…Finally, l1Fru from Rhodopseudomonas sphaeroides also appears to have at least one activity-linked cysteine, although the phosphorylation site has not yet been determined. Interestingly, this protein appears to bind Zn2+, and the binding of this cation protects against thiol oxidation and concomitant inactivation (268). The role if any, of this Zn2+-binding site in IIF'I remains to be determined.…”