Orotate Phosphoribosyltransferase-Mechanism

描述（由申请人提供）：酶具有独特的以高速率进行复杂化学反应的能力。为了做到这一点，他们在活性位点创造了一个化学环境，允许过渡态的形成。通常情况下，蛋白质本身含有移动元件，其运动对活性位点的形成至关重要，而这种运动与过渡状态发展的耦合是当代酶学家面临的一个重要问题。5-磷酸核糖基从其通用供体5-磷酸核糖基-1-焦磷酸（PRPP）转移到特定的杂环上形成核苷酸是由磷酸核糖基转移酶（PRTases）完成的。这些酶是遗传疾病的基因位点，激活抗肿瘤前药，是设计新的化疗药物的靶点。prtase分为两个进化组，每一个都是在研究者之前的研究中首次发现的。I型旋转PRTase （OMP合成酶）是本研究的主要靶点，它遵循Rossman折叠结构，在活性位点附近有一个突出的肽环，含有必需的催化残基。利用核磁共振定量分析了环运动与催化化学之间的联系，最近解决了所有底物结合的OMP合酶的环向下形式的三维结构。酶经过环和非环残基的重排序和结合底物的运动来实现催化。环闭合和化学之间的联系现在通过在环的每个残基中改变的具有动力学特征的突变OMP合成酶来更密切地探索。通过测定突变型OMP合酶的三维结构，以及野生型新产物和终端配合物的三维结构，揭示催化环和底物运动在催化中的结构作用。化学淬灭技术将被用于跟踪酶上化学和结合研究，以确定环改变对底物相互作用的影响。动力学同位素效应（KIE）技术和位置同位素交换（PIX）研究将测量结合底物在跨越过渡态势垒和解离之间的比例，以确定环闭合和过渡态形成是如何联系在一起的。该项目的第二个目标是II型喹啉酸PRTase，其一般机制尚不清楚。野生型酶的初始产物复合物的类似物的三维结构将被确定，PRPP和具有Arg 118突变体的喹啉酸盐类似物的复合物将被确定，其中两种底物之间的结合和两个亚基之间的连接已被破坏。KIE研究将确定QAPRTase的过渡态结构。

DESCRIPTION (provided by applicant): Enzymes are uniquely able to carry out difficult chemical reactions at high rates. To do this they create a chemical environment in the active site which allows for transition state formation. Frequently, the protein itself contains mobile elements whose motion is essential for active site formation, and coupling of this motion with transition state development is an important problem for the contemporary enzymologist. The transfer of the ribose 5-phosphate group from its universal donor, 5-phosphoribosyl-1-pyrophosphate (PRPP) to specific heterocycles to form nucleotides is carried out by the phosphoribosyltransferases (PRTases). These enzymes are loci of inherited disease, activate anti-tumor prodrugs, and are targets for design of new chemotherapeutics. The PRTases fall into two evolutionary groups, each first identified in the investigator's previous studies. The Type I orotate PRTase (OMP synthase), the major target of this work, follows Rossman fold architecture, and has a prominent peptide loop adjacent to the active site, containing essential catalytic residues. NMR was employed to quantitate the linkage between loop motion and catalytic chemistry and the three dimensional structure of a loop-down form of OMP synthase with all substrates bound has recently been solved. The enzyme undergoes a remarkable reordering of loop and non-loop residues and movement of bound substrates to achieve catalysis. The linkage between loop closing and chemistry is now explored more closely using kinetically characterized mutant OMP synthases altered in each residue of the loop. By determining the three-dimensional structures of mutant OMP synthases, and also of new products and dead-end complexes with wild type, the structural role of the catalytic loop and of substrate movement in catalysis will be revealed. Chemical quench techniques will be employed to follow on-enzyme chemistry, and binding studies to determine the effect of loop alteration on substrate interactions. Kinetic isotope effect (KIE) techniques and positional isotope exchange (PIX) studies will measure proportioning of bound substrates between crossing of the transition state barrier and dissociation to determine how loop closing and formation of the transition state are linked. A second target of the project is the Type II quinolinic acid PRTase, whose general mechanism is poorly understood. The three-dimensional structures of an analog of the initial products complex of wild-type enzyme will be determined as will a complex of PRPP and a quinolinate analog with an Arg 118 mutant in which linkage between binding of the two substrates and between the two subunits has been disrupted. KIE studies will determine the transition state structure of QAPRTase.