Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase
Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase
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DOI：
10.1074/jbc.m802866200
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发表时间：
2008-07-18
影响因子：
4.8
通讯作者：
Carman, George M.
中科院分区：
文献类型：
--
作者：
Han, Gil-Soo;O'Hara, Laura;Carman, George M.
酿酒酵母DGK 1基因编码一种催化二酰基甘油形成磷脂酸的二酰基甘油激酶。与来自细菌、植物和动物的二酰基甘油激酶不同，酵母酶利用CTP而不是ATP作为反应中的磷酸供体。Dgk 1 p含有CTP转移酶结构域，其存在于SEC 59编码的长胆醇激酶和CDS 1编码的CDP-二酰基甘油合酶中。缺失分析表明CTP转移酶结构域足以使甘油二酯激酶具有活性。CTP转移酶结构域内保守残基的点突变（R76 A、K77 A、D177 A和G184 A）导致二酰基甘油激酶活性丧失。DGK 1等位基因分析表明，Dgk 1 p在体内的功能，特别是由于它的二酰基甘油激酶活性。DGK 1编码的酶的最适pH为7.0-7.5，活性需要Ca 2（+）或Mg 2（+）离子，被N-乙基马来酰亚胺有效抑制，并且在40 ℃以上的温度下不稳定。该酶对甘油二酯（表观Km = 6.5mol%）表现出正协同动力学（Hill数= 2.5），对CTP（表观Km = 0.3mM）表现出饱和动力学。dCTP是酶的底物（表观Km = 0.4 mM）和竞争性抑制剂（表观Ki = 0.4 mM）。甘油二酯激酶活性的刺激主要膜磷脂和抑制CDP-甘油二酯和鞘氨醇碱。
The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2(+) or Mg2(+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent K-m = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K-m = 0.3mM). dCTP was both a substrate (apparent K-m = 0.4 mM) and competitive inhibitor (apparent K-i = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.