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• W2022201565 abstract "The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of Km values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated Km values (i.e. 5 to >300-fold). Each of these mutants displayed Vmax values that were reduced by ≥98% and resultant catalytic efficiencies that were reduced by ≥99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level. The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of Km values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated Km values (i.e. 5 to >300-fold). Each of these mutants displayed Vmax values that were reduced by ≥98% and resultant catalytic efficiencies that were reduced by ≥99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level. The mitochondrial citrate transport protein (CTP) 3The abbreviations used are: CTP, citrate transport protein; MOE, molecular operating environment; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide; R2, coefficient of determination; TMD, transmembrane domain. is located within the inner mitochondrial membrane and catalyzes an obligatory exchange of the dibasic form of tricarboxylic acids (e.g. citrate and isocitrate) for other tricarboxylic acids or in higher eukaryotes for dicarboxylic acids (e.g. malate and succinate) or phosphoenolpyruvate (1Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). Once in the cytoplasm, the transported citrate serves as the prime carbon source fueling fatty acid, triacylglycerol, and cholesterol biosyntheses (2Watson J.A. Lowenstein J.M. J. Biol. Chem. 1970; 245: 5993-6002Abstract Full Text PDF PubMed Google Scholar, 3Endemann G. Goetz P.G. Edmond J. Brunengraber H. J. Biol. Chem. 1982; 257: 3434-3440Abstract Full Text PDF PubMed Google Scholar, 4Brunengraber H. Lowenstein J.M. FEBS Lett. 1973; 36: 130-132Crossref PubMed Scopus (37) Google Scholar, 5Conover T.E. Trends Biochem. Sci. 1987; 12: 88-89Abstract Full Text PDF Scopus (35) Google Scholar). In addition, the concerted action of citrate lyase and malate dehydrogenase enables the generation of NAD+, a cofactor that is essential for the glycolytic pathway. Based on these roles, the CTP is considered essential for eukaryotic cell metabolism. Because of the prominent role of the CTP in cellular bioenergetics, our laboratory has conducted extensive investigations with the aim of elucidating its structure-based mechanism. Thus we have cloned (6Kaplan R.S. Mayor J.A. Wood D.O. J. Biol. Chem. 1993; 268: 13682-13690Abstract Full Text PDF PubMed Google Scholar), overexpressed (7Xu Y. Mayor J.A. Gremse D. Wood D.O. Kaplan R.S. Biochem. Biophys. Res. Commun. 1995; 207: 783-789Crossref PubMed Scopus (45) Google Scholar, 8Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and purified (9Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar, 10Eriks L.R. Mayor J.A. Kaplan R.S. Anal. Biochem. 2003; 323: 234-241Crossref PubMed Scopus (33) Google Scholar) the functional form of this transporter. Recently, employing a Cys-less yeast mitochondrial CTP construct that displays native functional properties (11Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) as the template, we have: (i) demonstrated that the transporter exists as a homodimer in detergent micelles (12Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. J. Bioenerg. Biomembr. 1999; 31: 543-549Crossref PubMed Scopus (38) Google Scholar); (ii) utilized cysteine-scanning mutagenesis combined with probing the accessibility of single-Cys mutants to MTS reagents, in both the absence and presence of citrate, to identify those residues in transmembrane domains III and IV that line the substrate translocation pathway (13Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Ma C. Kotaria R. Mayor J.A. Eriks L.R. Dean A.M. Walters D.E. Kaplan R.S. J. Biol. Chem. 2004; 279: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Ma C. Kotaria R. Mayor J.A. Remani S. Walters D.E. Kaplan R.S. J. Biol. Chem. 2005; 280: 2331-2340Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar); (iii) developed a detailed homology model of the three-dimensional structure of the CTP (16Walters D.E. Kaplan R.S. Biophys. J. 2004; 87: 907-911Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) based on the crystal structure of the mitochondrial ADP/ATP carrier (17Pebay-Peyroula E. Dahout-Gonzalez C. Kahn R. Trezeguet V. Lauquin J.-M.G Brandolin G. Nature. 2003; 426: 39-44Crossref PubMed Scopus (813) Google Scholar); and (iv) superimposed our functional data onto this homology model to delineate substantial portions of the translocation pathway within the structure (15Ma C. Kotaria R. Mayor J.A. Remani S. Walters D.E. Kaplan R.S. J. Biol. Chem. 2005; 280: 2331-2340Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 16Walters D.E. Kaplan R.S. Biophys. J. 2004; 87: 907-911Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). With this background in mind, the current studies focused on characterizing the kinetic properties of a panel of single-Cys mutants that we had previously constructed based on their predicted importance in the CTP mechanism (11Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 14Ma C. Kotaria R. Mayor J.A. Eriks L.R. Dean A.M. Walters D.E. Kaplan R.S. J. Biol. Chem. 2004; 279: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 18Ma C. Remani S. Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. Biochim. Biophys. Acta. 2006; 1757: 1271-1276Crossref PubMed Scopus (9) Google Scholar). We identified ten mutations that display both substantially elevated Km values and reduced Vmax values, nine of which localize to two topographically distinct clusters within the CTP structure. Modeling studies indicate that, although citrate is capable of numerous hydrogen bonding interactions with each cluster, one molecule of citrate cannot simultaneously interact with all members of both clusters. These findings have led us to propose a model, wherein the CTP contains at least two discrete substrate binding sites per monomer. The details of these sites and the implications of this model are discussed in detail. Construction, Overexpression, and Isolation of Single-Cys CTP Variants—Single-Cys CTP variants were prepared as previously described (14Ma C. Kotaria R. Mayor J.A. Eriks L.R. Dean A.M. Walters D.E. Kaplan R.S. J. Biol. Chem. 2004; 279: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, single-Cys CTP genes were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). Mutagenic primers were analyzed with Oligo 5.0 software. PCR amplifications and subsequent cloning steps were conducted according to the manufacturer's instructions. The Cys-less yeast mitochondrial CTP gene in pET-21a(+) was utilized as the starting template (11Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Transformants were screened for the presence of inserts via restriction digestion of plasmid DNA with NdeI and BamHI. The DNA from positive clones was then partially sequenced to assure the presence of the desired mutation. Plasmids scoring positively for the mutation of interest were then subcloned into the storage (Nova-Blue) and expression (BL21(DE3)) hosts. Mutations were confirmed by sequencing both strands of the entire CTP open reading frame. BL21(DE3) growth and induction of expression with isopropyl 1-thio-β-d-galactopyranoside (1 mm) were carried out according to the manufacturer's instructions (Novagen). Two hours following the addition of isopropyl 1-thio-β-d-galactopyranoside, three 200-ml aliquots of each culture were removed and placed on ice for 5 min. Following cell harvesting and resuspension, the three aliquots from each culture were combined into one tube. Cells were then lysed, and the inclusion body fraction was purified essentially as previously detailed (8Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The CTP was extracted by resuspension of the final inclusion body pellet in 6 ml of ice-cold 1.2% (w/v) Sarkosyl, which was dissolved in buffer, followed by centrifugation at 314,000 × g for 45 min. The supernatant contains the solubilized CTP. Protein was quantified via the method of Kaplan and Pedersen (19Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar). Incorporation of Single-Cys CTP Variants into Liposomal Vesicles—Overexpressed, Sarkosyl-solubilized, single-Cys CTP mutants were incorporated into liposomal vesicles, in the presence of 48 mm citrate, via the freeze-thaw sonication procedure as previously described (8Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 9Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar). Immediately prior to the transport assay, a given sample was thawed, sonicated on ice, and the extraliposomal citrate was removed via chromatography on a Dowex column in a Pasteur pipette. The sample was immediately used to assay transport. It should be noted that, with single-Cys mutants K83C, R87C, G119C, E122C, R181C, R189C, and R276C, whose Km values are much higher than those of many of the other mutants studied, the concentration of citrate included in the reconstitution incubation was increased to 75 mm. Following freeze-thaw sonication, the resulting higher level of extraliposomal citrate necessitated utilization of a larger capacity Dowex column for its removal. Thus, with these incubations, external citrate was removed on an Amersham Biosciences column containing 3 ml of Dowex beads. Determination of the Kinetic Parameters of CTP Variants before and after Modification with MTS Reagents—The kinetic parameters (Km and Vmax) of the Cys-less and the single-Cys CTP variants before modification with MTS reagents were determined as follows (15Ma C. Kotaria R. Mayor J.A. Remani S. Walters D.E. Kaplan R.S. J. Biol. Chem. 2005; 280: 2331-2340Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Proteoliposomes (45 μl) were preincubated with 3.5 μl of either buffer (experimental sample) (9Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar) or 200 mm 1,2,3-benzenetricarboxylate (control sample) for 10-12 min and were then further incubated with 3.5 μl of deionized/distilled water for an additional 10-12 min. The transport reaction was triggered via the addition of 21.5 μl of varying concentrations of [1,5-14C]citrate (Amersham Biosciences). Typically, ∼10 different external citrate concentrations bracketing the Km value were employed. However, with several CTP variants that displayed extremely high Km values (i.e. K83C, R87C, R189C, and R276C) substrate concentrations that were greater than the Km value could not be utilized because 50 mm external citrate was the highest concentration that we could employ and still maintain a specific radioactivity that was sufficiently high to enable accurate measurement of transport activity. Also, with very high Km mutants, the [14C]citrate source was concentrated by evaporation under vacuum prior to making up the stock solutions. Following transport incubations that ranged from 12 s to 2.5 h (depending on the intrinsic activity of a given CTP mutant), the experimental sample was quenched by the addition of 3.5 μl of 200 mm 1,2,3-benzenetricarboxylate. The control sample received an equal volume of buffer. Transport reactions were conducted at room temperature (21 °C). Following all incubations, intraliposomal radiolabeled citrate was separated from the external radiolabel via chromatography on short (i.e. 4 cm) Dowex columns in Pasteur pipettes. However, with those incubations containing a high external citrate concentration, we employed longer (i.e. 8 cm) Dowex columns to ensure the effective removal of extraliposomal citrate. The eluted (i.e. intraliposomal) radiolabel was quantified via liquid scintillation counting. The 1,2,3-benzenetricarboxylate-sensitive transport rate was calculated by subtracting the control value from the experimental value. The rate of uptake versus substrate concentration curves were fitted to the Michaelis-Menten equation, v = Vmax × S/(Km + S), using a non-linear least squares curve fit in GraphPad Prism. The final Km and Vmax values for each single-Cys CTP mutant, before and after modification with MTS reagents (see below), were calculated by taking the mean of the best fit Km and Vmax values derived from each separate V versus S profile. The measurement of the kinetic parameters following MTS labeling of the single-Cys mutants was conducted in a manner very similar to that described above. Consequently, only the methodological differences are described below. Proteoliposomes (45 μl) were preincubated with 3.5 μl of either buffer (experimental sample) or 200 mm 1,2,3-benzenetricarboxylate (control sample) for 10 min and were then further incubated with 3.5 μl of MTS reagent (each MTS reagent concentration in the reaction mix = 1.0 mm) for an additional 10 min. The remaining procedures were exactly as described above. Molecular Modeling of the CTP—Molecular modeling was carried out using version 5 of DOCK (20Shoichet B.K. Kuntz I.D. Bodian D.L. J. Comput. Chem. 1992; 13: 380-397Crossref Scopus (374) Google Scholar), obtained from the University of California at San Francisco, and version 2005.06 of Molecular Operating Environment (MOE), obtained from Chemical Computing Group, Montreal. The homology-modeled CTP structure was constructed as described previously (16Walters D.E. Kaplan R.S. Biophys. J. 2004; 87: 907-911Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Citrate, modeled in multiple conformations as a dianion, was initially docked into the CTP model using DOCK. In DOCK, five different scoring grids, centered at varying depths in the transport path, were utilized to ensure that all regions were thoroughly examined. The resulting docking poses were found to be clustered in two distinct regions of the translocation pathway. The first cluster was located about 10 Å into the pathway, centered around Lys-83, Arg-87, and Arg-189. The second cluster was located about 20 Å into the pathway, centered around Lys-37, Arg-181, Lys-239, Arg-276, and Arg-279. In the modeled CTP structure, these were found to be spread over too large an area to constitute a single binding site; even with side chains fully extended, the most distant residues are at least 20 Å apart. In contrast, a fully extended citrate molecule is <9 Å long. This result strongly suggested that there must be two citrate binding sites. We examined this possibility by mutating each of the above residues, as well as additional residues in the vicinity of these binding sites, to cysteine, one at a time. After kinetic analysis of these mutants verified the likely location of the above residues at a given citrate binding site, the two-binding site model was subjected to further refinement as follows. With all residues within 4.5 Å of the citrate, alternate side-chain conformations were explored using MOE. Models were then minimized using the CHARMM27 force field (21MacKerell Jr., A.D. Feig M. Brooks III, C.L. J. Comput. Chem. 2004; 25: 1400-1415Crossref PubMed Scopus (2879) Google Scholar) in MOE. For each cluster, the most energetically favorable orientation was selected, producing two final proposed binding sites as described below. Kinetic Characterization of a Panel of Single-Cys CTP Substitution Mutants Hypothesized to Participate in Substrate Binding and/or in Other Aspects of the Transport Mechanism—The present investigations focused on the kinetic characterization of a panel of 30 single-Cys substitution mutants that were previously constructed (11Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 14Ma C. Kotaria R. Mayor J.A. Eriks L.R. Dean A.M. Walters D.E. Kaplan R.S. J. Biol. Chem. 2004; 279: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 18Ma C. Remani S. Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. Biochim. Biophys. Acta. 2006; 1757: 1271-1276Crossref PubMed Scopus (9) Google Scholar) utilizing the Cys-less CTP as the template. Mutations of four groups of residues were characterized. The first group consisted of eight arginine and lysine residues, which, based on our molecular modeling results (see “Experimental Procedures”), cluster in two topographically distinct regions within the transport pathway. Residues Lys-83, Arg-87, and Arg-189 constitute one cluster and were predicted to form one binding site, and residues Lys-37, Arg-181, Lys-239, Arg-276, and Arg-279 comprise the second cluster and were predicted to form a second binding site. Each cluster appeared capable of forming multiple ionic hydrogen bonds with citrate. The second group consisted of residues that were predicted to reside nearby a given substrate binding site but were thought unlikely to be its prime determinants. Thus, Leu-116, Gly-119, Leu-120, Ser-123, Gln-182, Asn-185, and Gln-186 were predicted to be near binding site one and residues Glu-34, Phe-76, Glu-131, Lys-134, Thr-228, Val-229, Asp-236, Thr-240, and Gln-243 were predicted to be near binding site two. The effect of Cys substitution at several of these locations had been characterized previously as part of our studies into the role of TMDs III and IV in CTP function (15Ma C. Kotaria R. Mayor J.A. Remani S. Walters D.E. Kaplan R.S. J. Biol. Chem. 2005; 280: 2331-2340Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 18Ma C. Remani S. Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. Biochim. Biophys. Acta. 2006; 1757: 1271-1276Crossref PubMed Scopus (9) Google Scholar). The third group consisted of selected residues in matrix loops A (i.e. Thr-38, Arg-39, Asp-44, Lys-45, Ser-47, and Lys-48) and C (Asp-140, Lys-141, Gln-142, Ser-143, and Tyr-148). The rationale for exploring the role of these residues in CTP function was based upon findings with the mitochondrial ADP/ATP carrier (22Kihira Y. Hashimoto M. Shinohara Y. Majima E. Terada H. J. Biochem. 2006; 139: 575-582Crossref PubMed Scopus (5) Google Scholar, 23Hashimoto M. Shinohara Y. Majima E. Hatanaka T. Yamazaki N. Terada H. Biochim. Biophys. Acta. 1999; 1409: 113-124Crossref PubMed Scopus (65) Google Scholar), which suggested that, rather than project into the matrix compartment, these loops may in fact project into the translocation pathway during portions of the transport cycle and possibly assume an important role in the translocation mechanism. Finally, we characterized the mutation of Glu-122 because our previous studies indicated that this residue was essential for function. As depicted in Fig. 1 and Table 1, the mutated residues are located throughout the CTP structure.TABLE 1Effect of cysteine substitution on the CTP kinetic parametersMutationKmVmaxVmax/KmPredicted topographical locationnMnmol/min/mgnmol/min/mg/mM(%)Cys-less0.461 ± 0.0532305.0 ± 78.25000.0 (100)K83C>100NDaND, not determined.0.0 (0.0)TMD II, binding site oneR87C87.613 ± 58.863bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.0.2 ± 0.1cValues are p < 0.001.0.0 (0.0)TMD II, binding site oneG119C5.624 ± 1.224cValues are p < 0.001.0.3 ± 0.0cValues are p < 0.001.0.1 (0.0)TMD III, binding site oneR189C151.385 ± 59.715cValues are p < 0.001.2.4 ± 0.8cValues are p < 0.001.0.0 (0.0)TMD IV, binding site oneK37C2.817 ± 0.103cValues are p < 0.001.1.9 ± 0.1cValues are p < 0.001.0.7 (0.0)TMD I, binding site twoR181C24.085 ± 5.455cValues are p < 0.001.1.8 ± 0.1cValues are p < 0.001.0.1 (0.0)TMD IV, binding site twoK239C7.843 ± 2.405cValues are p < 0.001.43.3 ± 11.3cValues are p < 0.001.5.5 (0.1)TMD V, binding site twoR276C>100ND0.0 (0.0)TMD VI, binding site twoR279C3.721 ± 0.758cValues are p < 0.001.0.1 ± 0.0cValues are p < 0.001.0.0 (0.0)TMD VI, binding site twoE34C0.381 ± 0.03511.6 ± 0.3cValues are p < 0.001.30.4 (0.6)TMD I, near binding site twoT38C0.110 ± 0.018bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.307.2 ± 9.4cValues are p < 0.001.2792.7 (55.9)TMD I/Matrix Loop AR39C0.373 ± 0.0031732.0 ± 94.5dValues are p < 0.01.4643.4 (92.9)TMD I/Matrix Loop AD44C0.296 ± 0.065360.7 ± 11.8cValues are p < 0.001.1218.6 (24.4)Matrix Loop AK45C0.234 ± 0.030903.4 ± 25.4cValues are p < 0.001.3860.7 (77.2)Matrix Loop AS47C0.367 ± 0.0111102.0 ± 9.5cValues are p < 0.001.3002.7 (60.1)Matrix Loop AK48C0.297 ± 0.0421312.0 ± 63.5cValues are p < 0.001.4417.5 (88.4)Matrix Loop AF76C0.670 ± 0.194504.9 ± 2.2cValues are p < 0.001.753.6 (15.1)TMD II, near binding site twoL116C0.228 ± 0.008eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.487.1 ± 22.8cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.2136.4 (71.8)eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD III, near binding site oneL120C0.290 ± 0.014fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.455.5 ± 26.1cValues are p < 0.001.,fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.1570.7 (31.8)fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD III, near binding site oneE122C2.298 ± 0.764dValues are p < 0.01.1.7 ± 0.3cValues are p < 0.001.0.7 (0.0)TMD III, dimer interfaceS123C2.502 ± 0.928cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.103.4 ± 14.5cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.41.3 (1.4)eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD III, near binding site oneE131C0.170 ± 0.014bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.12.3 ± 0.3cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.72.4 (2.4)eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD III, near binding site twoK134C1.339 ± 0.168cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.55.8 ± 11.7cValues are p < 0.001.,eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.41.7 (1.4)eData adapted from Ref. 15. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD III, near binding site twoD140C0.064 ± 0.002dValues are p < 0.01.49.9 ± 2.9cValues are p < 0.001.779.7 (15.6)Matrix Loop CK141C0.716 ± 0.0021434.0 ± 12.5cValues are p < 0.001.2002.8 (40.1)Matrix Loop CQ142C0.304 ± 0.0161341.0 ± 101.5cValues are p < 0.001.4411.2 (88.2)Matrix Loop CS143C0.281 ± 0.034798.0 ± 31.8cValues are p < 0.001.2839.9 (56.8)Matrix Loop CY148C0.070 ± 0.000dValues are p < 0.01.26.4 ± 0.5cValues are p < 0.001.377.1 (7.5)Matrix Loop CQ182C0.162 ± 0.011dValues are p < 0.01.,fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.60.4 ± 3.7cValues are p < 0.001.,fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.372.8 (7.5)fData adapted from Ref. 18. The Vmax/Km percentage values were calculated for these mutants utilizing the control value presented in the cited publication.TMD IV, near binding site oneN185C0.578 ± 0.072372.8 ± 15.9cValues are p < 0.001.645.0 (12.9)TMD IV, near binding site oneQ186C0.302 ± 0.037109.8 ± 5.0cValues are p < 0.001.363.6 (7.3)TMD IV, near binding site oneT228C0.137 ± 0.019bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.431.8 ± 20.8cValues are p < 0.001.3151.8 (63.0)TMD V, near binding site twoV229C0.084 ± 0.005dValues are p < 0.01.174.7 ± 25.0cValues are p < 0.001.2079.8 (41.6)TMD V, near binding site twoD236C0.323 ± 0.04912.4 ± 0.3cValues are p < 0.001.38.4 (0.8)TMD V, near binding site twoT240C0.102 ± 0.036bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.53.4 ± 2.9cValues are p < 0.001.523.5 (10.5)TMD V, near binding site twoQ243C0.089 ± 0.001bValues are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.62.8 ± 1.7cValues are p < 0.001.705.6 (14.1)TMD V/E, near binding site twoa ND, not determined.b Values are p < 0.05 from a two-tailed Student's t test between Cys-less and individual single-Cys mutants.c Values are p < 0.001.d Values are p < 0.01.e Data adapted from Ref. 15Ma C. Kotaria R. Mayor J.A. Remani S. Walters D.E. Kaplan R.S. J. Biol. Chem" @default.
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• W2022201565 title "Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein" @default.
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