Orotate Phosphoribosyltransferase-Mechanism

描述(由申请人提供)：酶是唯一能够以高速率进行复杂化学反应的物质。为了做到这一点，他们在活性中心创造了一个化学环境，允许形成过渡态。通常，蛋白质本身含有运动元件，其运动对于活性位点的形成是必不可少的，而这种运动与过渡态发展的耦合是当代酶学家面临的一个重要问题。核糖5-磷酸基团从其通用供体5-磷酸核糖-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转移酶(PRTase)完成的。这些酶是遗传性疾病的基因座，激活抗肿瘤前药，是设计新的化疗药物的靶点。PRTase分为两个进化群体，每个群体都是在研究者之前的研究中首次发现的。I型orotate PRTase(OMP合成酶)是本工作的主要目标，遵循Rossman折叠结构，在活性部位附近有一个突出的肽环，含有必要的催化残基。利用核磁共振定量研究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环状OMP合成酶的三维结构。该酶经历了显着的环和非环残基的重新排序和结合底物的移动，以实现催化。现在使用在环的每个残基中改变的动力学特征的突变OMP合成酶来更密切地探索环关闭和化学之间的联系。通过测定突变的OMP合成酶以及新产物和野生型末端复合体的三维结构，将揭示催化环和底物运动在催化中的结构作用。化学猝灭技术将用于后续的酶化学和结合研究，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)研究将测量结合底物在跨越过渡态势垒和解离之间的比例，以确定环关闭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体的类似物的三维结构将被确定，PRPP和喹啉类似物与Arg 118突变体的复合体也将被确定，在该复合体中，两种底物之间的结合和两个亚基之间的连接被破坏。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.