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 carnitine acyltransferase I (CAT I) regulate the entry of fatty acyl moieties into their respective organelles. Thus, CPT I and CAT I occupy prominent positions in the pathways responsible for energy generation in mitochondria and the assembly of VLDL in the endoplasmic reticulum, respectively. Previous attempts to determine the intrinsic kinetic properties of CPT I and CAT I have been hampered by the occurrence of sigmoidal velocity curves. This was overcome, in this study, by the inclusion of recombinant acyl-CoA binding protein in the assay medium. For the first time, we have determined the concentrations 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 docosahexaenoic acid (C22:6). The data show that carnitine inhibits CAT I at physiological concentrations which are not inhibitory to CPT I. Thus, carnitine concentration is likely to be a significant factor in determining the partitioning of acyl-CoAs between mitochondria and microsomes, a role which has not been previously recognized. Moreover, the finding that CAT I elicits a lower 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 polyunsaturated fatty acid to inhibit VLDL biosynthesis.
Abo-Hashema
K A
KA
Schools of Biological Sciences & Biotechnology and Mathematical & Physical Sciences, Division of Science and Engineering, Murdoch University, Murdoch, W.A. 6150, Australia.
Cake
M H
MH
Lukas
M A
MA
Knudsen
J
J
D000214:Acyl Coenzyme A / Q000737:chemistry; D002331:Carnitine / Q000032:analysis; D002333:Carnitine Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; C044663:docosahexaenoyl-coenzyme A; C017585:oleoyl-coenzyme A; D002333:Carnitine Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine