Drosophila alcohol dehydrogenase:: Acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis
Drosophila alcohol dehydrogenase:: Acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis
复制标题
DOI：
10.1016/j.jmb.2004.10.028
复制
发表时间：
2005-01-21
影响因子：
5.6
通讯作者：
Ladenstein, R
中科院分区：
文献类型：
--
作者：
Benach, J;Winberg, JO;Ladenstein, R
果蝇乙醇脱氢酶 (DADH) 是一种 NAD(+) 依赖性酶，可催化醇氧化为醛/酮，并且还能够进一步将醛氧化为其相应的羧酸。通过 X 射线晶体学以 1.6 埃的分辨率解析了黑腹果蝇 ADH 的慢速等位基因 (DmADH-S) 的三元酶-NADH-乙酸复合物的结构。醛歧化反应的辅酶立体化学表明，虽然没有发生酶促反应，但所获得的酶-NADH-乙酸复合物反映了高效的三元复合物。分叉底物结合位点中乙酸酯结合的立体化学，以及之前醛还原和醇氧化的立体化学研究表明，醛的甲基在还原反应中与 R1 结合，在氧化反应中与 R2 子位点结合。 NMR 研究以及之前的动力学研究表明，乙醇氧化为乙酸时形成的乙醛中间体在还原型辅酶之前离开底物位点，然后与新形成的酶-NAD(+) 复合物结合。在这里，我们比较了黑腹果蝇 ADH-S 的三维结构和之前理论上建立的模型，评估了五种黎巴嫩果蝇 ADH 的晶体结构在多种复杂形式中的差异，解释了底物特异性以及这两种酶基于晶体结构的微妙动力学差异。我们还重新研究了蛋白质表面带电残基的静电对酶催化效率的影响。 (C) 2004 Elsevier Ltd. 保留所有权利。
Drosophila alcohol dehydrogenase (DADH) is an NAD(+)-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 Angstrom resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD(+) complex. Here, we compare the three-dimensional structure of D. melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme. (C) 2004 Elsevier Ltd. All rights reserved.