Nicot Carine, Relat Joana, Woldegiorgis Gebre, Haro Diego, Marrero Pedro F
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
J Biol Chem. 2002 Mar 22;277(12):10044-9. doi: 10.1074/jbc.M109976200. Epub 2002 Jan 14.
Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras.
猪和大鼠肝脏肉碱棕榈酰转移酶I(L-CPTI)对棕榈酰辅酶A和肉碱具有相同的K(m)值。然而,它们对丙二酰辅酶A抑制作用的敏感性差异很大。因此,猪L-CPTI对丙二酰辅酶A的IC(50)为141纳米,而大鼠L-CPTI的IC(50)为2微摩尔。通过构建大鼠L-CPTI和猪L-CPTI之间的嵌合体,我们发现整个C末端区域表现为一个单一结构域,它决定了该酶对丙二酰辅酶A的整体敏感性。丙二酰辅酶A敏感性的程度由该结构域所采用的结构决定。通过缺失突变分析,我们发现丙二酰辅酶A敏感性还取决于这个单一结构域与N末端前18个氨基酸残基的相互作用。我们得出结论,猪和大鼠L-CPTI具有不同的丙二酰辅酶A敏感性,因为N末端前18个氨基酸残基与C末端结构域的相互作用方式不同。这是第一项利用活性C末端嵌合体描述C末端和N末端区域之间的相互作用如何决定L-CPTI酶对丙二酰辅酶A敏感性的研究。