Department of Molecular Medicine, University of South Florida, Tampa, Florida 33612, USA.
J Biol Chem. 2010 Dec 3;285(49):38104-14. doi: 10.1074/jbc.M110.169102. Epub 2010 Sep 17.
DHHC protein acyltransferases (PATs) catalyze the palmitoylation of eukaryotic proteins through an enzymatic mechanism that remains largely unexplored. In this study we have combined genetic and biochemical approaches to examine the molecular mechanism of palmitate transfer of the yeast Ras PAT, which is composed of Erf2 and Erf4. The palmitoylation reaction consists of two steps; they are autopalmitoylation of the enzyme to create a palmitoyl-Erf2 intermediate followed by the transfer of the palmitoyl moiety to the Ras substrate. Palmitoyl-CoA serves as the palmitate donor. To elucidate the kinetic properties of the Erf2·Erf4 PAT, we have developed a coupled enzyme assay that monitors the turnover of the palmitoyl-enzyme species indirectly by measuring the rate of CoASH release. Mutational analysis indicates that the DHHC motif constitutes the catalytic core of the enzyme required for autopalmitoylation and palmitoyl transfer to the Ras2 substrate. In the absence of Ras2, the palmitoyl-Erf2·Erf4 complex undergoes a cycle of hydrolysis and re-palmitoylation, implying that in the presence of palmitoyl-CoA, the complex is autopalmitoylated and competent to transfer palmitate to a protein substrate.
DHHC 蛋白酰基转移酶 (PAT) 通过酶促机制催化真核蛋白的棕榈酰化，而该机制在很大程度上仍未得到探索。在这项研究中，我们结合了遗传和生化方法来研究酵母 Ras PAT 的棕榈酸转移的分子机制，该酶由 Erf2 和 Erf4 组成。棕榈酰化反应由两个步骤组成；它们是酶的自身棕榈酰化，以创建棕榈酰-Erf2 中间体，然后将棕榈酰部分转移到 Ras 底物上。棕榈酰-CoA 作为棕榈酸供体。为了阐明 Erf2·Erf4 PAT 的动力学特性，我们开发了一种偶联酶测定法，通过测量 CoASH 释放的速率间接监测棕榈酰酶物种的周转。突变分析表明，DHHC 基序构成了酶的催化核心，对于酶的自身棕榈酰化和棕榈酰转移到 Ras2 底物是必需的。在没有 Ras2 的情况下，棕榈酰-Erf2·Erf4 复合物经历水解和再棕榈酰化的循环，这意味着在棕榈酰-CoA 的存在下，该复合物进行自身棕榈酰化，并能够将棕榈酸转移到蛋白质底物上。
