Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A
Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A
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DOI：
10.1074/jbc.m700885200
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发表时间：
2007-06-22
影响因子：
4.8
通讯作者：
Gomez-Puertas, Paulino
中科院分区：
文献类型：
--
作者：
Lopez-Vinas, Eduardo;Bentebibel, Assia;Gomez-Puertas, Paulino
在L肉碱存在下，肉毒碱棕榈酰转移酶1催化棕榈酰辅酶A转化为棕榈酰肉碱，从而促进脂肪酸进入线粒体，这一过程是被丙二酰辅酶A生理抑制的。为了研究丙二酰辅酶A抑制CPT1肝脏异构体(CPT1A)的机制，我们构建了其NH2-和COOH-末端结构域的电子模型。发现两个丙二酰辅酶A结合位点。其中“CoA位点”或“A位点”参与了NH2-和COOH-末端结构域之间的相互作用，并共享酰基-CoA半隧道。另一个是“CoA相反位”或“O位”，在酶的另一边，在催化通道中。这两个位点共享肉碱结合基因。为了防止NH2-和COOH-末端区域之间的相互作用，我们产生了CPT1AE26K和K561E突变体。还产生了一个双突变体E26K/K561E(SWAP)，该突变体有望保护相互作用。抑制实验表明，CPT1AE26K和K561E单一突变体对丙二酰辅酶A的敏感性(IC50)降低了12倍，而SWAP突变体恢复到野生型IC50值。我们得出结论，两个结构域之间的结构相互作用对于酶对“A位”丙二酰辅酶A抑制的敏感性至关重要。表达的R243T突变体的抑制实验证实了丙二酰辅酶A结合的“O位”的位置。该模型也被动力学实验所支持，该实验表明丙二酰辅酶A对肉碱具有线性混合型抑制作用。丙二酰辅酶A改变肉碱的亲和力，肉碱Km与丙二酰辅酶A IC50呈指数负相关。
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of L-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.