Evaluation of the affinity and turnover number of both hepatic mitochondrial and microsomal carnitine acyltransferases: Relevance to intracellular partitioning of acyl-CoAs

Mitochondrial carnitine palmitoyltransferase I(CPT I) and microsomal carnit ine acyltransferase I (CAT I) regulate the entry of fatty acyl moieties int o their respective organelles. Thus, CPT I and CAT I occupy prominent posit ions in the pathways responsible for energy generation in mitochondria and the assembly of VLDL in the endoplasmic reticulum, respectively. Previous a ttempts to determine the intrinsic kinetic properties of CPT I and CAT I ha ve been hampered by the occurrence of sigmoidal velocity curves. This was o vercome, in this study, by the inclusion of recombinant acyl-CoA binding pr otein in the assay medium. For the first time, we have determined the conce ntrations of total functional enzyme (E-t) by specific radiolabeling of the active site, the dissociation constants (K-d) and the turnover numbers of CPT I and CAT I toward the CoA esters of oleic acid (C18:1) and docosahexae noic acid (C22:6). The data show that carnitine inhibits CAT I at physiolog ical concentrations which are not inhibitory to CPT I. Thus, carnitine conc entration is likely to be a significant factor in determining the partition ing of acyl-CoAs between mitochondria and microsomes, a role which has not been previously recognized. Moreover, the finding that CAT I elicits a lowe r turnover toward the CoA ester of C22:6 (25 s(-1)) than toward that of C18 :1 (111 s(-1)), while having similar K-d values, suggests the use of this p olyunsaturated fatty acid to inhibit VLDL biosynthesis.