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J. Biol. Chem., Vol. 282, Issue 25, 18212-18224, June 22, 2007

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Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A^*

Eduardo López-Viñas^||1, Assia Bentebibel^||1, Chandrashekaran Gurunathan^||, Montserrat Morillas^||2, Dolores de Arriaga^¶, Dolors Serra^||, Guillermina Asins^||, Fausto G. Hegardt^||3, and Paulino Gómez-Puertas^||

From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, E-28049 Madrid, Spain, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain, ^¶Departamento de Biología Molecular, Universidad de León, E-24071 León, Spain, and ^||CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of L-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH₂- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH₂- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH₂- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC₅₀) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC₅₀ value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine K_m and malonyl-CoA IC₅₀.

Received for publication, January 30, 2007 , and in revised form, April 17, 2007.

^* This study was supported by Dirección General de Investigación Científica y Técnica, Spain, Grants BMC2001-3048 and SAF2004-06843; Ajut de Suport als Grups de Recerca de Catalunya Grant 2005SGR-00733 (to F. G. H.); and a grant from Fundación Ramón Areces (to the Centro de Biología Molecular "Severo Ochoa"). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ Both authors contributed equally to this work.

² Present address: Dept. de Ciencias Experimentales y de la Salud, Universidad Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, E-08003 Barcelona, Spain.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, Diagonal 643, E-08028 Barcelona, Spain. E-mail: fgarciaheg{at}ub.edu.

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