Morillas Montserrat, Gómez-Puertas Paulino, Bentebibel Assia, Sellés Eva, Casals Nuria, Valencia Alfonso, Hegardt Fausto G, Asins Guillermina, Serra Dolors
Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, E-08028 Barcelona, Spain.
J Biol Chem. 2003 Mar 14;278(11):9058-63. doi: 10.1074/jbc.M209999200. Epub 2002 Dec 23.
Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ariño, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.
肉碱棕榈酰转移酶（CPT）I催化棕榈酰辅酶A转化为棕榈酰肉碱，促进其通过线粒体膜运输，它受到丙二酰辅酶A的抑制。通过使用SequenceSpace算法程序来识别所有肉碱酰基转移酶中参与丙二酰辅酶A抑制作用的氨基酸，我们发现了5个保守氨基酸（苏氨酸（314）、天冬酰胺（464）、丙氨酸（478）、甲硫氨酸（593）和半胱氨酸（608），以大鼠肝脏CPT I的坐标为准），这些氨基酸存在于可被抑制的丙二酰辅酶A酰基转移酶（肉碱辛酰转移酶和CPT I）中，而在不可被抑制的丙二酰辅酶A酰基转移酶（CPT II、肉碱乙酰转移酶（CAT）和胆碱乙酰转移酶（ChAT））中不存在。为了确定这些氨基酸残基在丙二酰辅酶A抑制中的作用，我们制备了五重突变体CPT I T314S/N464D/A478G/M593S/C608A以及五个单突变体CPT I T314S、N464D、A478G、M593S和C608A。在每种情况下，所选择的CPT I氨基酸都被突变为CPT II、CAT和ChAT中相同同源位置的氨基酸。由于突变体M593S几乎消除了对丙二酰辅酶A的敏感性，因此制备了另外两个甲硫氨酸（593）突变体：M593A和M593E。突变体A478G和C608A以及所有甲硫氨酸（593）突变体中CPT I以肉碱为底物的催化效率（V(max)/K(m)）明显提高。在那些甲硫氨酸（593）已发生突变的CPT I蛋白中，对丙二酰辅酶A的敏感性几乎消除。丙氨酸（478）、半胱氨酸（608）和苏氨酸（314）突变为CPT II、CAT和ChAT中的同源氨基酸残基会导致丙二酰辅酶A敏感性出现不同程度的降低。丙氨酸（478）位于CPT I的结构模型中靠近催化位点的位置，并参与低亲和力位点中丙二酰辅酶A的结合（莫里利亚斯，M.，戈麦斯 - 普尔塔斯，P.，鲁比，B.，克洛泰特，J.，阿里尼奥，J.，巴伦西亚，A.，赫加德特，F.G.，塞拉，D.，和阿辛斯，G.（2002年）《生物化学杂志》277，11473 - 11480）。甲硫氨酸（593）可能参与第二个亲和力位点中丙二酰辅酶A的相互作用，该位点的位置尚未报道。
