Arg-425 of the citrate transporter CitP is responsible for high affinity binding of di- and tricarboxylatese

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes excha nge of divalent anionic citrate from the medium for monovalent anionic lact ate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was th e subject of this investigation. Previous studies indicated that CitP is sp ecific for substrates containing a 2-hydroxycarboxylate motif, HO-CR2-COO-. CitP has a high affinity for substrates that have a "second" carboxylate a t one of the R groups, such as divalent citrate and (S)-malate (Bandell, M. , and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anion ic substrates that lack this second carboxylate were found to bind with a l ow affinity, In the present study we have constructed site-directed mutants , changing Arg-425 into a lysine or a cysteine residue. By using two substr ates, Be. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-ma late was strongly decreased, whereas the affinity for monovalent 2-hydroxyi sobutyrate was not. The largest effect was seen when the arginine was chang ed into the neutral cysteine, which reduced the affinity for (S)-malate ove r 50-fold. Chemical modification of the R425C mutant with the sulfhydryl re agent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425 C and R425K mutants revealed a substrate protectable inhibition by other su lfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, r espectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates , and thus plays an important role in the generation of the membrane potent ial.