COMPARISON OF THE ACTIVE-SITES OF THE PURIFIED CARNITINE ACYLTRANSFERASES FROM PEROXISOMES AND MITOCHONDRIA BY USING A REACTION-INTERMEDIATE ANALOG

The carnitine acyltransferases contribute to the modulation of the acy l-CoA/CoA ratio in various cell compartments with consequent effects o n many aspects of fatty acid metabolism. The properties of the enzymes are different in each location. The kinetic mechanisms and kinetic pa rameters for the carnitine acyltransferases purified from peroxisomes (COT) and from the mitochondrial inner membrane (CPT-II) were determin ed. Product-inhibition studies established that COT follows a rapid-eq uilibrium random-order mechanism, but CPT-II follows a strictly ordere d mechanism in which acyl-CoA or CoA must bind before the carnitine su bstrate. Hemipalmitoylcarnitinium [(+)-HPC], a prototype tetrahedral i ntermediate analogue of the acyltransferase reaction, inhibits CPT-II 100-feud better than COT. (+)-HPC behaves as an analogue of palmitoyl- L-carnitine with COT. In contrast, with CPT-II (+)-HPC binds more tigh tly to the enzyme than do substrates or products, suggesting that it i s a good model for the transition state and, unlike palmitoyl-L-carnit ine, (+)-HPC can bind to the free enzyme. The data support the concept of three binding domains for the acyltransferases, a CoA site, an acy l site and a carnitine site. The CoA site is similar in COT and CPT-II , but there are distinct differences between the carnitine-binding sit e which may dictate the kinetic mechanism.