HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 50, 39130-39136, December 15, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/50/39130    most recent M005940200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bandell, M. Articles by Lolkema, J. S. Search for Related Content PubMed PubMed Citation Articles by Bandell, M. Articles by Lolkema, J. S. Social Bookmarking                      What's this?

Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates^*

Michael Bandell and Juke S. Lolkema^§

From the Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands

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-CR₂-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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				W. A. M. Wolken, P. M. Lucas, A. Lonvaud-Funel, and J. S. Lolkema The Mechanism of the Tyrosine Transporter TyrP Supports a Proton Motive Tyrosine Decarboxylation Pathway in Lactobacillus brevis J. Bacteriol., March 15, 2006; 188(6): 2198 - 2206. [Abstract] [Full Text] [PDF]

				I. Sobczak and J. S. Lolkema The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism Microbiol. Mol. Biol. Rev., December 1, 2005; 69(4): 665 - 695. [Abstract] [Full Text] [PDF]

				I. Sobczak and J. S. Lolkema Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae J. Biol. Chem., July 23, 2004; 279(30): 31113 - 31120. [Abstract] [Full Text] [PDF]

				B. P. Krom, R. Aardema, and J. S. Lolkema Bacillus subtilis YxkJ Is a Secondary Transporter of the 2-Hydroxycarboxylate Transporter Family That Transports L-Malate and Citrate J. Bacteriol., October 15, 2001; 183(20): 5862 - 5869. [Abstract] [Full Text] [PDF]