Yonetani-Theorell analysis of hepatic carnitine palmitoyltransferase-I inhibition indicates two distinct inhibitory binding sites.

Analysis of inhibitor studies indicates that carnitine palmitoyltransferase-I has two separate sites for inhibitor binding. One site is located on the cytoplasmic side of the mitochondrial outer membrane. Malonyl-CoA, the most important physiological inhibitor of carnitine palmitoyltransferase-I, binds primarily to this site, but it can also bind to another site. A second inhibitory site is located at the active site of carnitine palmitolytransferase-I. Coenzyme A, a product/inhibitor of carnitine palmitoyltransferase-I binds primarily at this site and can inhibit carnitine palmitoyltransferase-I at physiological concentrations, but can also attenuate malonyl-CoA inhibition. Analogs of malonyl-CoA and other simpler compounds containing two carbonyl groups but no coenzyme A moiety inhibit only at the cytoplasmic site, indicating that this site has an absolute requirement for two carbonyl groups but has no absolute requirement for a coenzyme A moiety. Inhibitors acting through the active site included the active-site-directed inhibitor (+)-hemipalmitoylcarnitinium. These studies support the existence of two regulatory binding sites for the control of hepatic fatty acid oxidation: (a) the active site, for regulation by the inhibitory binding of coenzyme A and acetyl-CoA, and (b) a separate regulatory malonyl-CoA-binding site that is physical separated from the active site.