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 μM. 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 μM). 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 μM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K_I of 6 μM 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 Mg²⁺, with the EC₅₀ for inhibition being 3 mM free Mg²⁺. The low affinity triacsin C inhibition was also enhanced by Mg²⁺. The data suggests that Mg²⁺ 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 Mg²⁺. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:100–106, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20009