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 shortchain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 muM. 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 muM). 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 muM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K-I of 6 muM 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+. (C) 2004 Wiley Periodicals, Inc.