Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transpo rt of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mut ant variant from rat peroxisomal COT were expressed in Saccharomyces cerevi siae. Both expressed forms were expressed similarly in quantitative terms a nd exhibited full enzyme activity. The wild-type-expressed COT was inhibite d by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 mu M). These results indicate that the two hist idine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from, which it is concluded that it plays an important role in catalysis. ( C) 2000 Federation of European Biochemical Societies.