Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A.
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 Km and malonyl-CoA IC50.
López-Viñas
Eduardo
E
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, E-28049 Madrid, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
Bentebibel
Assia
A
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
Gurunathan
Chandrashekaran
C
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
Morillas
Montserrat
M
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
de Arriaga
Dolores
D
Departamento de Biología Molecular, Universidad de León, E-24071 León, Spain.
Serra
Dolors
D
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
Asins
Guillermina
G
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
Hegardt
Fausto G
FG
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain. Electronic address: fgarciaheg@ub.edu.
Gómez-Puertas
Paulino
P
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, E-28049 Madrid, Spain; CIBER Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28049 Madrid, Spain.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002331:Carnitine / Q000737:chemistry; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine