Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria.

Abstract

Phosphate/2-deoxyglucose 6-phosphate antiport in Streptococcus lactis showed an exchange stoichiometry that varied over a 2-fold range when assay pH was shifted between pH 8.2 and pH 5.2. At pH 7.0 and above, 2 mol of phosphate moved per mol of sugar phosphate; at pH 6.1 the ratio was 1.5:1, while at pH 5.2 the overall stoichiometry fell to 1. (Fig. LA) represented about a 100-fold accumulation of substrate above its original medium concentration. The vesicles, loaded with sugar phosphate, were then reisolated by centrifugation, resuspended, and redistributed into three tubes, having assay buffer at pH 7.0, pH 6.1, or pH 5.2.Abbreviation: Mops, 3-(N-morpholino)propanesulfonic acid. 280The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Ins and Outs of Major Facilitator Superfamily Antiporters

“…For technical reasons discussed by Maloney et al (53), investigations into the stoichiometry of glucose-6-phosphate(G6P)/phosphate exchange were performed on membrane vesicles of S. lactis. A striking finding from this work was the effect of pH on the substrate-exchange process (9,52). First, a reduction in pH slowed the reaction due to a velocity effect.…”

“…Dropping the pH from 7 to 5.2 reduced the V max 10-fold, whereas the K m remained constant for both heterologous and homologous exchange reactions, implying that monobasic and dibasic sugars were equally effective as substrates. Second, the pH had a direct impact on stoichiometry, with a 2:1 P i :sugar phosphate exchange measured at pH 7, and a 1:1 exchange measured at pH 5.2 (9). Based on these findings, along with the presumption that monovalent P i but not divalent P i is the preferred exchange substrate, a model was forwarded that incorporated a bifunctional substrate binding site that accepted either two monovalent sugar phosphates (2HG6P 1-) or a single divalent anion (1G6P 2-), but not both (53).…”

Transmembrane Segment 11 of UhpT, the Sugar Phosphate Carrier ofEscherichia coli, Is an α-Helix That Carries Determinants of Substrate Selectivity

“…Early work indicated that the exchange reactions mediated by UhpT are electrically neutral in nature (27,54), and the abrupt alteration in selectivity that accompanies lysine insertion at positions 388 or 391 reinforces the idea that maintenance of electrostatic neutrality is an essential criterion. We also believe that this same view can help interpret the behavior of the K391C variant, which shows a distinct acid shift in the pH optimum for growth and transport (Figs.…”

Secondary solute transport in bacteria

“…At pH 7.0 (0.9 pH units above the pK 2 of glucose 6-phosphate) the carrier catalyzes exchange of two molecules of monovalent phosphate for one molecule of divalent glucose 6-phosphate, whereas at pH 5.2 (0.9 pH units below the pK 2 of glucose 6-phosphate) the exchange corresponds with one molecule of monovalent phosphate for one molecule of monovalent glucose 6-phosphate. This aspect, however, has best been documented for the phosphate/sugar 6-phosphate antiporter of L. lactis [87], but is most likely also true for UhpT. (vii) UhpT requires a rather high ionic strength (0.3 M KC1) for maximal activity, a phenomenon that has not yet been explained.…”

“…Both forces thus work together which allows a high rate of exchange. The Ap or one of its components can affect the translocation process through (de)protonation of the substrate(s) and/or through the differential charge of the individual substrates, e.g., sugar-phosphate/phosphate [87], malate/lactate [43], oxalate/ formate [82], and lysine/alanine exchange [88]. Some systems catalyze precursor/product exchange or solute-H + symport, depending on the concentrations of the solutes and protons on either side of the membrane, the dissociation constants (K o) for these molecules and the magnitude of the membrane potential, e.g., lactose/galactose exchange in S. thermophilus [89], arginine/ornithine exchange in Pseudomonas aeruginosa [90], malate/lactate exchange in L. lactis [43].…”