Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues.
Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues.
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肉毒碱棕榈酰转移酶 I 亚型的 N 端和 C 端结构域在酶的丙二酰辅酶 A 敏感性中的作用：嵌合蛋白表达和保守组氨酸残基突变的见解。
DOI：
10.1042/bj3350513
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Brown,NF
中科院分区：
文献类型：
--
作者：
Swanson,ST;Foster,DW;McGarry,JD;Brown,NF
线粒体外膜酶肉碱棕榈酰基转移酶I （CPT I）通过丙二酰辅酶a的抑制作用，在脂肪酸进入线粒体基质进行β-氧化的调控中起主要作用。CPT I的两种亚型，肝脏型(L)和肌肉型(M)，已被确定，后者对丙二酰辅酶a的敏感性高100倍，对底物肉碱的km高得多。在这里，我们研究了CPT I分子的不同区域在它们对丙二酰辅酶a、依托莫西（一种不可逆抑制剂）和肉毒碱的反应中的作用。为此，我们分析了工程大鼠CPT I构建物的特性，其中(a) L-CPT I的n端结构域被删除，(b) L-和M-CPT I的n端结构域被切换，或(c)位于L-CPT I n端的三个保守组氨酸残基中的每一个都发生突变。出现了几个新的观点：(1)尽管n端结构域对正常的丙二酰辅酶a反应至关重要，但它本身并不能解释肝脏和肌肉酶对抑制剂的广泛不同的敏感性；(2) His-5和/或His-140可能在丙二醇- coa反应中起直接作用，但His-133不起作用；(3)该酶的截短型、嵌合型和点突变型变体均与共价的、活性位点导向的配体依托莫西结合；(4)只有L-CPT I最根本的改变，即n端82个残基的缺失，才会影响对肉毒碱的反应。我们得出结论，CPT I的n端结构域在丙二酰辅酶a对酶的抑制中起着重要但允许的作用。相比之下，较大的c端区域决定了对丙二酰辅酶a的敏感程度，以及对肉碱的反应；它也足以结合依托莫西。此外，进一步增加了一个或多个组氨酸残基可能参与CPT -丙二醇-辅酶a相互作用的概念。
The mitochondrial outer membrane enzyme carnitine palmitoyltransferase I (CPT I) plays a major role in the regulation of fatty acid entry into the mitochondrial matrix for β-oxidation by virtue of its inhibition by malonyl-CoA. Two isoforms of CPT I, the liver type (L) and muscle type (M), have been identified, the latter being 100 times more sensitive to malonyl-CoA and having a much higherKmfor the substrate carnitine. Here we have examined the roles of different regions of the CPT I molecules in their response to malonyl-CoA, etomoxir (an irreversible inhibitor) and carnitine. To this end, we analysed the properties of engineered rat CPT I constructs in which (a) the N-terminal domain of L-CPT I was deleted, (b) the N-terminal domains of L- and M-CPT I were switched, or (c) each of three conserved histidine residues located towards the N-terminus in L-CPT I was mutated. Several novel points emerged: (1) whereas the N-terminal domain is critical for a normal malonyl-CoA response, it does not itself account for the widely disparate sensitivities of the liver and muscle enzymes to the inhibitor; (2) His-5 and/or His-140 probably play a direct role in the malonyl-CoA response, but His-133 does not; (3) the truncated, chimaeric and point- mutant variants of the enzyme all bound the covalent, active-site- directed ligand, etomoxir; and (4) only the most radical alteration of L-CPT I, i.e. deletion of the N-terminal 82 residues, affected the response to carnitine. We conclude that the N-terminal domain of CPT I plays an essential, but permissive, role in the inhibition of the enzyme by malonyl-CoA. By contrast, the larger C-terminal region dictates the degree of sensitivity to malonyl-CoA, as well as the response to carnitine; it is also sufficient for etomoxir binding. Additionally, further weight is added to the notion that one or more histidine residues may be involved in the CPT I–malonyl-CoA interaction.