Hepatic carnitine palmitoyltransferase-I has two independent inhibitory binding sites for regulation of fatty acid oxidation

Khosrow Kashfi, Randall L. Mynatt, George Cook

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Partial proteolysis of carnitine palmitoyltransferase (CPT-I) in intact mitochondria results in greatly diminished sensitivity to inhibition by its physiological inhibitor, malonyl-CoA, but inhibition by succinyl-CoA and methylmalonyl-CoA was affected to a lesser extent, whereas inhibition by coenzyme A, acetyl-CoA, and propionyl-CoA was not affected at all by proteinase treatment. These data suggested that inhibitors that are coenzyme A esters of short-chain dicarboxylic acids bind to a regulatory malonyl-CoA binding site located on the cytoplasmic face of the mitochondrial outer membrane while coenzyme A esters of monocarboxylic acids and free coenzyme A act at the active site in the mitochondrial intermembrane space. All inhibitors whose potency was altered by proteinase action provided protection against proteinases, whereas other inhibitors did not. Preincubation with the substrates carnitine, palmitoyl-CoA, or coenzyme A prior to proteolysis showed no protective effects against the loss of inhibition or loss of activity; however, preincubation with these substrates enhanced proteinase effects to more seriously diminish activity and inhibition by malonyl-CoA. Proteinases were also found to act on purified mitochondrial outer membranes to reduce inhibition by malonyl-CoA with little effect on activity. Using these outer membrane preparations it was found that the very potent inhibition of CPT-I by the active-site-directed substrate analog (+)-hemipalmitoylcarnitinium was not altered by proteinase treatment; however, inhibition by the malonyl-CoA analog Ro 25-0187, which is a more potent inhibitor than malonyl-CoA, was drastically reduced by proteinase treatment of mitochondrial outer membranes, confirming the different locations for the malonyl-CoA site and the active site. Coenzyme A and malonyl-CoA both act as competitive inhibitors with respect to the acyl-CoA substrate, but coenzyme A lacks cooperative effects seen with malonyl-CoA. For ligand binding to the malonyl-CoA regulatory site, there appears to be a requirement for two carbonyl groups in close juxtaposition, but there is apparently no requirement for the coenzyme A moiety per se. Current evidence, including the recently deduced primary structure for CPT-I, favors the hypothesis that (a) inhibitors of CPT-I may act at two distinct sites, (b) malonyl-CoA binds primarily to a regulatory site that is distinct from the active site of carnitine palmitoyltransferase-I, and (c) the two inhibitory sites are located on opposite sides of the mitochondrial outer membrane.

Original languageEnglish (US)
Pages (from-to)245-252
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume1212
Issue number2
DOIs
StatePublished - May 13 1994

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Malonyl Coenzyme A
Carnitine O-Palmitoyltransferase
Fatty Acids
Coenzyme A
Binding Sites
Oxidation
Liver
Peptide Hydrolases
Mitochondrial Membranes
Catalytic Domain
Membranes
Proteolysis
Substrates
Esters
Palmitoyl Coenzyme A
Dicarboxylic Acids
Acyl Coenzyme A
Acetyl Coenzyme A
Mitochondria
Carnitine

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Endocrinology

Cite this

Hepatic carnitine palmitoyltransferase-I has two independent inhibitory binding sites for regulation of fatty acid oxidation. / Kashfi, Khosrow; Mynatt, Randall L.; Cook, George.

In: Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism, Vol. 1212, No. 2, 13.05.1994, p. 245-252.

Research output: Contribution to journalArticle

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