Orotate Phosphoribosyltransferase-Mechanism

描述（由申请人提供）：酶是唯一能够以高速率进行困难化学反应的酶。为了做到这一点，它们在活性部位创造了一个化学环境，允许过渡态的形成。通常，蛋白质本身含有移动的元素，它们的运动对于活性位点的形成是必不可少的，这种运动与过渡态发展的耦合是当代酶学家的一个重要问题。核糖5-磷酸基团从其通用供体5-磷酸核糖基-1-焦磷酸（PRPP）转移到特定杂环以形成核苷酸是通过磷酸核糖基转移酶（PRTases）进行的。这些酶是遗传性疾病的基因座，激活抗肿瘤前药，并且是新化疗药物设计的靶标。PRTases分为两个进化组，每一个都是在研究者以前的研究中首次发现的。I型乳清酸PRTase（OMP合成酶），这项工作的主要目标，遵循罗斯曼折叠结构，并具有一个突出的肽环相邻的活性位点，含有必需的催化残基。NMR被用来定量环运动和催化化学之间的联系，最近已经解决了与所有底物结合的OMP合酶的环下形式的三维结构。该酶经历环和非环残基的显著重排以及结合底物的移动以实现催化。环闭合和化学之间的联系，现在探索更密切地使用动力学特征的突变体OMP酶改变的每个残基的循环。通过确定突变OMP酶的三维结构，以及新产品和野生型的死端复合物的三维结构，将揭示催化环和底物运动在催化中的结构作用。将采用化学淬灭技术跟踪酶化学和结合研究，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）研究将测量过渡态势垒跨越和解离之间的结合底物比例，以确定环闭合和过渡态形成之间的联系。该项目的第二个目标是II型喹啉酸PRTase，其一般机制知之甚少。将测定野生型酶的初始产物复合物的类似物的三维结构，如PRPP和喹啉类似物与Arg 118突变体的复合物，其中两种底物的结合之间的连接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.