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Adv Exp Med Biol. 1999;466:27-42.

Topology of hepatic mitochondrial carnitine palmitoyltransferase I.

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Department of Physical Sciences, Kingsborough Community College, City University of New York, Brooklyn, USA.


Our earlier work using intact mitochondria and isolated mitochondrial outer membranes confirms the observations of Murthy and Pande that CPT-I is located on the mitochondrial outer membranes and supports the notion that this enzyme has a malonyl-CoA binding domain facing the cytosol and an acyl-CoA binding domain facing the inter membrane space. Our data also suggests that coenzyme A binds at the active site of CPT-I, as does acyl-CoA, 2-bromopalmitoyl-CoA, and (+)-hemipalmitoylcarnitinium, but malonyl-CoA does not bind at that site. Inhibition of CPT-I at the malonyl-CoA binding site by HPG and Ro 25-0187, which have no CoA moiety, contributes to a resolution of this question in that the CoA itself is not essential for the binding of malonyl-CoA to its regulatory site, but the dicarbonyl function which is present in malonyl-CoA, HPG, and Ro 25-0187 is absolutely essential. Our re-evaluation of the topology of hepatic mitochondrial CPT-I confirms the original observations that this enzyme has at least two different binding domains, one domain binding malonyl-CoA, HPG, and Ro-25-187 and the other domain binding acyl-CoA and other inhibitors of CPT-I. Furthermore, the malonyl-CoA binding domain is exposed to the cytosolic face of the membrane. Our data showing that treatment of the intact mitochondria with trypsin causes release of adenylate kinase which indicates that trypsin has damaged the mitochondrial outer membrane, possibly allowing trypsin to enter the intermembrane space and act on CPT from within the outer membrane. Since trypsin's action is limited to arginine and lysine residues, an alternative explanation could be that the portion of the protein domain responsible for malonyl-CoA inhibition may not contain these residues. The latter explanation is plausible, since malonyl-CoA was able to protect against loss of activity and sensitivity to inhibition, but did not protect against loss of adenylate kinase, suggesting that rupture of the outer membrane is not necessarily related to loss of CPT activity. These results suggest that some protein domain that is necessary for CPT activity is exposed on the outer surface of the outer membranes. Therefore, it seems likely that trypsin would have to be able to hydrolyse protein domains of CPT that are inaccessible to Nagarse and papain.

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