Structural mechanism of enoyl-CoA hydratase: Three atoms from a single water are added in either an E1cb stepwise or concerted fashion
Structural mechanism of enoyl-CoA hydratase: Three atoms from a single water are added in either an E1cb stepwise or concerted fashion
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DOI：
10.1021/bi015844p
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发表时间：
2002-02-26
期刊：
影响因子：
2.9
通讯作者：
Petsko, GA
中科院分区：
文献类型：
--
作者：
Bahnson, BJ;Anderson, VE;Petsko, GA
我们已经确定了酶的晶体结构的烯酰辅酶A水合酶（ECH）从大鼠肝脏与结合底物4-（N，N-二甲基氨基）肉桂酰基-CoA，使用X-射线衍射数据至2.3埃的分辨率除了硫醇酯底物之外，在水合反应中加入的催化水，已经被建模为晶体学不对称单元内生理六聚体的六个活性位点中的每一个中的明确定义的电子密度。催化水桥接酶的Glu（144）和Glu（164），并且具有孤对电子，准备与酶结合的α，β-不饱和硫酯的C反应。连接两个谷氨酸残基的水分子使人联想到烯醇化酶的活性位点。然而，与烯醇化酶不同，烯醇化酶具有可用于提供质子的赖氨酸，ECH中的其他活性位点残基没有其他质子来源。此外，对活性位点的氢键网络的分析表明，Glu（144）和Glu（164）都是离子化的，并且携带负电荷，没有合理的位置来具有质子化的羧酸盐。这种缺乏氢键受体可以容纳质子的来源，而不是来自水分子，导致一种假设，即来自单个水分子的三个原子被添加到双键上以形成水合产物。的结构结果进行了讨论，与详细的机制，这已阐明从动力学，定点诱变，和光谱的酶底物物种，在提出一个原子分辨率的反应机制。与先前的解释相反，E-S络合物的结构连同先前确定的动力学同位素效应与协调机制或E1 cb逐步机制一致。
We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 Angstrom In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.