Structural Basis for Bridged Bimetallic Enzyme Catalysis

总体目标是了解酶活性位点的环境如何控制由桥接配体连接的含有两个金属离子的催化中心的反应性。桥接双金属中心出现在数百种不同的酶中，催化几十种不同的反应，包括DNA、RNA和磷脂的降解和合成；氢化物离子转移；磷酰基转移；糖的水解；多肽的共价修饰和n端加工。然而，为什么要使用两种金属离子，以及它们如何在催化中合作，目前还不清楚。具体目标集中在：i)金属离子如何合作，ii)为什么特定的金属/配体组合在催化特定类型的反应中最有效，以及iii)活性位点的其余部分如何参与并帮助指导催化的化学反应。本研究选取了四种具有代表性的酶。在木糖异构酶中，桥接的Mg2+-Mg2+中心将葡萄糖转化为果糖，这是一个具有巨大商业意义的反应。氨肽酶在外肽酶反应中使用Zn2+-Zn2+中心；该酶超家族的成员是抗血管生成癌症药物的靶标。DRAG利用Mg2+-Mg2+催化共价修饰蛋白水解adp -核糖。免疫抑制剂靶点钙调磷酸酶在信号转导中使用Fe3+-Zn2+中心作为必需的丝氨酸/苏氨酸磷酸酶。我们的实验计划是基于这样的假设：桥接双金属酶利用其双核位点上的金属离子路易斯酸来：i)结合并定位底物，ii)结合并激活水分子以产生活性位点的氢氧亲核试剂，iii)作为“超亲电试剂”使底物上的化学键极化，从而促进催化反应过渡态的形成。我们将采用多种方法，包括我们自己开发的方法。这些技术包括蛋白质晶体学（常规、超高分辨率和时间分辨）、中子劳厄晶体学、酶动力学、光谱学（合作）、量子力学/分子力学计算和定点诱变。

The overall aim is to understand how the environment of an enzyme active site controls the reactivity of a catalytic center containing two metal ions connected by a bridging ligand. Bridged bimetallic centers appear in hundreds of different enzymes catalyzing dozens of different reactions including the degradation and synthesis of DNA, RNA and phospholipids; hydride ion transfer; phosphoryl group transfer; the hydrolysis of sugars; and the covalent modification and N-terminal processing of polypeptides. Yet why two metal ions are employed, and how they cooperate in catalysis is not understood. The specific aims focus on: i) how the metal ions cooperate, ii) why particular metal/ligand combinations are most effective in catalyzing particular types of reactions, and iii) how the rest of the active site participates in, and helps to direct the chemistry of, catalysis. Four representative enzymes have been selected for this study. In xylose isomerase a bridged Mg2+-Mg2+ center converts glucose to fructose, a reaction of enormous commercial importance. Aminopeptidase uses a Zn2+-Zn2+ center in an exopeptidase reaction; members of this enzyme superfamily are targets for antiangiogenic cancer drugs. DRAG uses Mg2+-Mg2+ to catalyze the hydrolysis of ADP-ribose from a covalently-modified protein. And the immunosuppressant target calcineurin uses an Fe3+-Zn2+ center in its role as an essential protein Ser/Thr phosphatase in signal transduction. Our experimental plan is guided by the hypothesis that bridged bimetallic enzymes use the metal ion Lewis acidities in their binuclear sites to: i) bind and position substrate, ii) bind and activate a water molecule to yield an active site hydroxide nucleophile, and iii) act as a "superelectrophile" to polarize a chemical bond on the substrate and thereby promote formation of the transition state of the catalytic reaction. We will employ a variety of methods, including ones we have developed ourselves. These techniques include protein crystallography (conventional, ultra-high resolution, and time-resolved), neutron Laue crystallography, enzyme kinetics, spectroscopy (in collaboration), quantum mechanics/molecular mechanics calculations, and site-directed mutagenesis.