Active-site-directed inhibition of carnitine acetyltransferase.
Methoxycarbonyl-CoA disulfide has been used as an active-site-directed inhibitor of carnitine acetyltransferase. Stoichiometric addition of methoxycarbonyl-CoA disulfide to carnitine acetyltransferase showed the modification of one sulfhydryl group with concomitant loss of about 80% enzyme activity. The rate of modification of this sulfhydryl group is an order of magnitude faster than that of the remaining sulfhydryl groups in the enzyme. Methoxycarbonyl-CoA disulfide inactivation is biphasic: k1 = 1.09 X 10(2) M-1 S-1, k2 = 1.1 X 10(1) M-1 S-1. This modification, Enz-SS-CoA is covalent; it can be reversed with either dithioerythritol or thiocholine. Acetyl-carnitine and acetyl-CoA protected the enzyme against methoxycarbonyl-CoA disulfide inactivation; however, carnitine did not. These results indicate the presence of a sulfhydryl group in carnitine acetyltransferase at the site of acetyl group transfer. Titration of carnitine acetyltransferase with nonspecific sulfhydryl reagents, DTNB, and rho-nitrophenoxycarbonyl methyl disulfide, revealed that four sulfhydryl groups were preferentially modified by these reagents. The results also show that seven other sulfhydryl groups are available for modification.
D000123:Acetyltransferases / Q000037:antagonists & inhibitors*; D001665:Binding Sites / Q000187:drug effects; D002332:Carnitine O-Acetyltransferase / Q000037:antagonists & inhibitors*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003065:Coenzyme A / Q000031:analogs & derivatives / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D013438:Sulfhydryl Compounds
D004220:Disulfides; D013438:Sulfhydryl Compounds; C022029:methoxycarbonyl-coenzyme A disulfide; C023778:4-nitrophenoxycarbonyl methyl disulfide; D004228:Dithionitrobenzoic Acid; D000123:Acetyltransferases; D002332:Carnitine O-Acetyltransferase; D003065:Coenzyme A