PT  - JOURNAL ARTICLE
AU  - Sreevidya Aluvila
AU  - Jiakang Sun
AU  - David H. T. Harrison
AU  - D. Eric Walters
AU  - Ronald S. Kaplan
TI  - Inhibitors of the Mitochondrial Citrate Transport Protein: Validation of the Role of Substrate Binding Residues and Discovery of the First Purely Competitive Inhibitor
AID  - 10.1124/mol.109.058750
DP  - 2010 Jan 01
TA  - Molecular Pharmacology
PG  - 26--34
VI  - 77
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/77/1/26.short
4100  - http://molpharm.aspetjournals.org/content/77/1/26.full
SO  - Mol Pharmacol2010 Jan 01; 77
AB  - The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (Kic) of 0.12 ± 0.02 mM and an uncompetitive inhibition constant (Kiu) of 3.04 ± 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the Kic values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the Km for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, Km approximates the Kd (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport. © 2010 The American Society for Pharmacology and Experimental Therapeutics