Morillas Montserrat, Gómez-Puertas Paulino, Rubí Blanca, Clotet Josep, Ariño Joaquín, Valencia Alfonso, Hegardt Fausto G, Serra Dolors, Asins Guillermina
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.
J Biol Chem. 2002 Mar 29;277(13):11473-80. doi: 10.1074/jbc.M111628200. Epub 2002 Jan 14.
Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.
肉碱辛酰转移酶（COT）和肉碱棕榈酰转移酶（CPT）I可促进中链和长链脂肪酸穿过过氧化物酶体膜和线粒体膜，它们在生理上受到丙二酰辅酶A的抑制。我们采用“计算机模拟”大分子对接方法构建了一个模型，其中丙二酰辅酶A可附着在催化核心附近。这扰乱了在报道的两种蛋白质模型中酰基辅酶A底物在通道中的定位（莫里拉斯，M.，戈麦斯 - 普尔塔斯，P.，罗卡，R.，塞拉，D.，阿辛斯，G.，巴伦西亚，A.，和赫加特，F.G.（2001年）《生物化学杂志》276卷，45001 - 45008页）。推测的丙二酰辅酶A结构域包含His(340)，其与His(131)一起与COT对丙二酰辅酶A的敏感性有关（莫里拉斯，M.，克洛泰特，J.，鲁比，B.，塞拉，D.，阿辛斯，G.，阿里尼奥，J.，和赫加特F.G.（2000年）《欧洲生物化学学会联合会快报》466卷，183 - 186页）。当我们将COT的His(131)突变时，IC(50)增加，并且丙二酰辅酶A与底物癸酰辅酶A竞争。COT的Ala(332)位于距His(340) 8个氨基酸的结构域中，其突变降低了COT对丙二酰辅酶A的敏感性。L - CPT I的同源组氨酸和丙氨酸残基His(277)、His(483)和Ala(478)也发生了突变，这降低了对丙二酰辅酶A的敏感性。在一名患有人类L - CPT I遗传性缺陷的患者中，同样位于丙二酰辅酶A预测位点的Pro(479)自然突变为Leu，改变了对丙二酰辅酶A的敏感性。我们得出结论，这个丙二酰辅酶A结构域存在于COT和L - CPT I蛋白中，可能是丙二酰辅酶A与底物酰基辅酶A相互作用的位点。该家族中其他对丙二酰辅酶A不敏感的成员，CPT II和肉碱乙酰转移酶，不包含这个结构域。
