Abstract
The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, 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 the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-). CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. 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 potential.
Highlights
In recent years a growing number of secondary transporters have been discovered that generate rather than consume metabolic energy
A recent study of chimeras between the citrate transporter citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter MleP indicated that the C-terminal region including transmembrane segments (TMSs) XI forms part of the substrate-binding site that interacts with the R groups of the substrates [8]
Construction and Activity of the R425C and R425K Mutant Transporters—Arg-425 is conserved in the transporters of the 2-hydroxycarboxylate transporter (2-HCT) family and located in the C-terminal putative TMS XI (Fig. 1)
Summary
2-HIB, 2-hydroxyisobutyrate; RSO, right-side-out; pCMB, p-chloromercuribenzoic acid; pCMBS, p-chloromercuribenzosulfonate; TNBS, 2,4,6-trinitrobenzene sulfonate; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; TMS, transmembrane segment; 2-HCT, 2-hydroxycarboxylate transporter. The C-terminal region contains two conserved arginine residues, one of which is located in TMS XI. The latter residue was thought to be a good candidate for an interaction with the second carboxylate of divalent substrates. It is concluded that Arg-425 is located in the substrate-binding site where it is responsible for a high affinity interaction with the second carboxylate of di- and tricarboxylates
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