Swanson S T, Foster D W, McGarry J D, Brown N F
Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9135, USA.
Biochem J. 1998 Nov 1;335 ( Pt 3)(Pt 3):513-9. doi: 10.1042/bj3350513.
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 beta-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 higher Km for 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.
线粒体外膜酶肉碱棕榈酰转移酶I（CPT I）通过丙二酰辅酶A对其的抑制作用，在调节脂肪酸进入线粒体基质进行β氧化过程中发挥着主要作用。已鉴定出CPT I的两种同工型，即肝型（L）和肌肉型（M），后者对丙二酰辅酶A的敏感性高100倍，且对底物肉碱的米氏常数（Km）高得多。在此，我们研究了CPT I分子不同区域在其对丙二酰辅酶A、依托莫司（一种不可逆抑制剂）和肉碱反应中的作用。为此，我们分析了工程化大鼠CPT I构建体的特性，其中（a）L-CPT I的N端结构域被删除，（b）L-CPT I和M-CPT I的N端结构域被交换，或（c）L-CPT I中位于N端的三个保守组氨酸残基中的每一个都发生了突变。出现了几个新观点：（1）虽然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酶和肌肉酶对抑制剂的广泛不同敏感性；（2）His-5和/或His-140可能在丙二酰辅酶A反应中起直接作用，但His-133不起作用；（3）该酶的截短、嵌合和点突变变体都结合了共价、活性位点导向配体依托莫司；（4）只有L-CPT I最彻底的改变，即删除N端的82个残基，才会影响对肉碱的反应。我们得出结论，CPT I的N端结构域在丙二酰辅酶A对该酶的抑制中起重要但非决定性作用。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；它对于依托莫司结合也足够了。此外，一个或多个组氨酸残基可能参与CPT I-丙二酰辅酶A相互作用这一观点得到了进一步支持。
