Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver.
Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site. The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).
Vessey
Donald A
DA
Department of Veterans' Affairs Medical Center, San Francisco, CA 94121, USA. donald.vessey@med.va.gov
Kelley
Michael
M
Warren
Robert S
RS
D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology; D014226:Triazenes / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D014226:Triazenes; C034613:triacsin C; D003066:Coenzyme A Ligases; D008274:Magnesium