Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA.
The active site of the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria was blocked by the self-catalysed formation of the S-carboxypalmitoyl-CoA ester of (-)-carnitine, followed by washing of the mitochondria. CPT I activity in treated mitochondria was inhibited by 90-95%. Binding of [14C]malonyl-CoA to these mitochondria was not inhibited as compared with that of control mitochondria. When CPT I activity was inhibited, palmitoyl-CoA could markedly displace [14C]malonyl-CoA binding from the low-affinity site for the inhibitor [Zammit, Corstorphine & Gray (1984) Biochem. J. 222, 335-342], but not from the high-affinity site for malonyl-CoA binding. The saturation characteristics of the malonyl-CoA-binding component lost in the presence of palmitoyl-CoA were sigmoidal, and thus suggestive of co-operative binding at this site. It is suggested that the site hitherto considered to be a low-affinity malonyl-CoA-binding site may be effectively a second, allosteric, acyl-CoA-binding site on CPT I under conditions that prevail in vivo, whereas the high-affinity site for malonyl-CoA may be exclusive to the inhibitor. The possibility that the competitive-type interactions of malonyl-CoA and acyl-CoA on CPT I activity could arise from the effects of separate malonyl-CoA and acyl-CoA allosteric sites is considered. The possible significance of the large difference in the capacity of the two sites and their different saturation kinetics is also discussed.
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002331:Carnitine / Q000494:pharmacology; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003065:Coenzyme A / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D051381:Rats
D000214:Acyl Coenzyme A; D007527:Isoenzymes; D010171:Palmitoyl Coenzyme A; D008316:Malonyl Coenzyme A; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine; D003065:Coenzyme A