Inhibitors of the Mitochondrial Citrate Transport Protein: Validation of the Role of Substrate Binding Residues and Discovery of the First Purely Competitive InhibitorS
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 competitiv...
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Veröffentlicht in: | Molecular pharmacology 2010-01, Vol.77 (1), p.26-34 |
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Sprache: | eng |
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Zusammenfassung: | 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 (
K
ic
) of 0.12 ± 0.02 mM
and an uncompetitive inhibition constant (
K
iu
) of 3.04
± 0.74 mM]. Based on docking calculations, a model for BTC binding has
been developed. We then determined the
K
ic
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
K
m
for citrate
transport with each of these binding site mutants, thereby confirming that with these
CTP variants,
K
m
approximates the
K
d
(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. |
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ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.109.058750 |