@article {Aluvilamol.109.058750, author = {Sreevidya Aluvila and Jiakang Sun and David H.T. Harrison and D. Eric Walters and Ronald S Kaplan}, title = {Inhibitors of the Mitochondrial Citrate Transport Protein: Validation of the Role of Substrate Binding Residues and Discovery of the First Purely Competitive Inhibitor}, elocation-id = {mol.109.058750}, year = {2009}, doi = {10.1124/mol.109.058750}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classical 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 {\textpm} 0.02 mM and an uncompetitive inhibition constant, Kiu of 3.04 {\textpm} 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 62-261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. Importantly, 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 therefore is 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 where the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) prior to transport.The American Society for Pharmacology and Experimental Therapeutics}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2009/10/20/mol.109.058750}, eprint = {https://molpharm.aspetjournals.org/content/early/2009/10/20/mol.109.058750.full.pdf}, journal = {Molecular Pharmacology} }