Four-dimensional structure analysis of Ndx family enzyme utilizing a new pH-temperature jump trigger

在开始我们的研究之前，我们试图提高Ndx家族adp -核糖焦磷酸酶（ADPRase）的晶体质量，因为我们需要高分辨率的分析来分辨在酶反应过程中ADPRase反应腔中共存的底物和产物。测试了许多结晶条件，并将可观察的衍射极限从先前报道的1.7A扩展到1.1A分辨率。为了启动ADPRase的晶态反应，将Zn（II）离子浸泡在晶体中，并在晶体中预先引入底物ADPR。由于温度从295K跳到90K，反应停止了。在0、3、6、10、15、20、30、40、60min 9个反应时间点测定晶体结构，并成功进行ADPRase反应的原位观察。结果表明，锌（II）离子浸泡后，酶在晶体中的反应进展如下：(1)第一个引入反应腔的Zn（II）离子使ADPR构象变为中间态，称为ADPR^*。(2)第二个Zn（II）离子连接了ADPR^*、E82和E86的gultamate侧链以及两个水分子，形成了五配体配位结构。(3)两个水分子中的一个被第二个Zn（II）离子和E82活化为氢氧根阴离子，(4)阴离子亲核攻击ADPR的α磷酸，使焦磷酸键断裂，生成两个产物AMP和核糖-5′-磷酸。讨论了原位观察ADPRase的生化意义。

Before starting our research, we tried to improve crystal quality of ADP-ribose pyro-phosphatase (ADPRase) involved in Ndx family, because we needed high resolution analysis to resolve substrates and products which would co-exist in the reaction cavity of ADPRase in time course of enzyme reaction. Many crystallization conditions were tested, and the observable diffraction limit was expanded to 1.1A resolution from 1.7A, previously reported. In order to start the crystalline state reaction of ADPRase, Zn(II) ions were soaked into the crystals, in which the substrate ADPR was introduced in advance. The reaction was stopped by a temperature jump from 295K to 90K. Crystal structures were determined at 9 points of reaction time, 0, 3, 6, 10, 15, 20, 30, 40, 60min, and the in situ observation of ADPRase reaction was succeeded. Our results showed that the enzyme reaction progressed in crystal after the Zn(II) ion soaking as following. (1) The first Zn(II) ion introduced into reaction cavity changed ADPR conformation to an intermediate state, designated as ADPR^*.(2) The second Zn(II) ion ligated ADPR^*, gultamate side chain of E82 and E86, and two water molecules making a five-ligands coordination structure. (3) One of two water molecules was activated by the second Zn(II) ion and E82 to hydroxide anion, and (4) the anion attacked nucleophilically the alpha phosphate of ADPR to brake the pyrophosphate bond to produce two products, AMP and ribose-5'-phosphate. The biochemical insight into the in situ observation of ADPRase was discussed.