Redesign of carnitine acetyltransferase specificity by protein engineering
Redesign of carnitine acetyltransferase specificity by protein engineering
复制标题
DOI：
10.1074/jbc.m402685200
复制
发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Serra, D
中科院分区：
文献类型：
--
作者：
Cordente, AG;Lopez-Viñas, E;Serra, D
在真核生物中，L-肉碱通过促进脂肪酸的β-氧化而参与能量代谢。肉毒碱乙酰转移酶（CrAT）催化乙酰辅酶A和肉毒碱可逆转化为乙酰肉毒碱和游离辅酶A。为了重新设计大鼠CrAT对其底物的特异性，我们突变了Met（564）。M564 G突变的CrAT对长链酰基辅酶A表现出更高的活性：对肉豆蔻酰辅酶A的活性是野生型CrAT的1250倍，而对其天然底物乙酰辅酶A的活性较低。动力学常数的突变体CrAT显示修改有利于较长的酰基辅酶A作为底物。在相反的情况下，肉毒碱辛酰转移酶（COT）中的邻位甘氨酸（Gly（553））突变为甲硫氨酸，降低了其天然底物（中链和长链酰基辅酶A）的活性，并增加了短链酰基辅酶A的活性。另一种CrAT突变体M564 A以相同的方式制备和测试，具有类似的结果。我们得出结论，Met（564）阻断了中链和长链酰基辅酶A进入CrAT的催化位点。野生型和突变的CrAT和COT的三维模型支持这一假设。我们首次表明，一个单一的氨基酸是能够确定CrAT和COT的底物特异性。
In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met(564). The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly(553)) to methionine in carnitine octanoyltransferase ( COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met(564) blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.