HEPATIC CARNITINE PALMITOYLTRANSFERASE-I HAS 2 INDEPENDENT INHIBITORYBINDING-SITES FOR REGULATION OF FATTY-ACID OXIDATION

Partial proteolysis of carnitine palmitoyltransferase (CPT-I) in intac t mitochondria results in greatly diminished sensitivity to inhibition by its physiological inhibitor, malonyl-CoA, but inhibition by succin yl-CoA and methylnalonyl-CoA was affected to a lesser extent, whereas inhibition by coenzyme A, acetyl-CoA, and propionyl-CoA was not affect ed at all by proteinase treatment. These data suggested that inhibitor s that are coenzyme A esters of short-chain dicarboxylic acids bind to a regulatory malonyl-CoA binding site located on the cytoplasmic face of the mitochondrial outer membrane while coenzyme A esters of monoca rboxylic acids and free coenzyme A act at the active site in the mitoc hondrial intermembrane space. All inhibitors whose potency was altered by proteinase action provided protection against proteinases, whereas other inhibitors did not. Preincubation with the substrates carnitine , palmitoyl-CoA, or coenzyme A prior to proteolysis showed no protecti ve effects against the loss of inhibition or loss of activity; however , preincubation with these substrates enhanced proteinase effects to m ore seriously diminish activity and inhibition by malonyl-CoA. Protein ases were also found to act on purified mitochondrial outer membranes to reduce inhibition by malonyl-CoA with little effect on activity. Us ing these outer membrane preparations it was found that the very poten t inhibition of CPT-I by the active-site-directed substrate analog (+) -hemipalmitoylcarnitinium was not altered by proteinase treatment; how ever, inhibition by the malonyl-CoA analog Ro 25-0187, which is a more potent inhibitor than malonyl-CoA, was drastically reduced by protein ase treatment of mitochondrial outer membranes, confirming the differe nt locations for the malonyl-CoA site and the active site. Coenzyme A and malonyl-CoA both act as competitive inhibitors with respect to the acyl-CoA substrate, but coenzyme A lacks cooperative effects seen wit h malonyl-CoA. For ligand binding to the malonyl-CoA regulatory site, there appears to be a requirement for two carbonyl, groups in close ju xtaposition, but there is apparently no requirement for the coenzyme A moiety per se. Current evidence, including the recently deduced prima ry structure for CPT-I, favors the hypothesis that (a) inhibitors of C PT-I may act at two distinct sites, (b) malonyl-CoA binds primarily to a regulatory site that is distinct from the active site of carnitine palmitoyltransferase-I, and (c) the two inhibitory sites are located o n opposite sides of the mitochondrial outer membrane.