Structure and Mechanism of Yeast Orotate PRTase

描述（由申请人提供）：迄今为止的证据清楚地表明， 酵母乳清酸磷酸核糖基转移酶的Theorell-Chance机理 （OPRTase），负责所有嘧啶的从头产生的酶 所有生物体中的核苷酸。动力学研究建议把这个 假设到严格的测试。主要研究者 完善了过表达系统， 获得重组天然酶。 他已经获得了晶体， 并将继续进行旨在生产衍射质量晶体的实验， 该酶与OPRT酶的适当配体复合。而且他 将尝试使酶与以下物质的感受态底物类似物共结晶： PRPP，已经由主要研究者合成，并与类似物， 其模拟过渡态的所涉及的氧代碳正离子性质。等 当然，类似物可以预期作为生物医学活性剂。 结晶实验的成功将允许， 合作研究，OPRTase的X射线结构详细说明了结合 沿着各种底物的基序以及所述底物的结构图。 这是由这种重要的酶催化的反应的过渡态。这样的 分析将与对动力学机制的理解齐头并进。 过渡态类似物也将被评价为各种抗氧化剂的抑制剂。 PRTases。他还提出了几种突变体（单突变体）的动力学评价， 或在活性位点利用位点处或附近的双氨基酸取代 定向诱变）形式的酵母OPRT酶，其被设计为具有减少的 稳定氧代碳正离子过渡态的能力。 预计这项研究将揭示新的机制， 关键酶的作用。一般来说，机械/结构研究，如 这些通常为抗代谢物的合理设计提供了强有力的手段- 可能是抗癌剂。

DESCRIPTION (provided by applicant): Evidence to date clearly points to a Theorell-Chance mechanism for yeast orotate phosphoribosyltransferase (OPRTase), the enzyme responsible for the de novo production of all pyrimidine nucleotides in all organisms. Kinetic studies are proposed to put this hypothesis to a rigorous test. The principal investigator has perfected an overexpression system that allows large quantities of the recombinant native enzyme to be obtained. He has obtained crystals that diffract to 6A and will continue experiments aimed at producing diffraction-quality crystals of this enzyme complexed with appropriate ligands for OPRTase. Additionally, he will attempt to co-crystallize the enzyme with competent substrate analogs of PRPP, already synthesized by the principal investigator, and with analogs, which mimic the implicated oxocarbocation nature of the transition-state. Such analogs may, of course, be expected to act as biomedically active agents. Success in the crystallization experiments will allow, in collaborative studies, (an) X-ray structure(s) of OPRTase detailing the binding motif(s) of the various substrate(s) along with a structural map of the transition-state of the reaction catalyzed by this important enzyme. Such an analysis will go hand in hand with an understanding of the kinetic mechanism. The transition state analogs would also be evaluated as inhibitors of various PRTases. He also proposes the kinetic evaluation of the several mutant (single or double amino acid substitution(s) at or near the active site utilizing site directed mutagenesis) versions of yeast OPRTase designed to have reduced capacity to stabilize an oxocarbocation transition-state. It is expected that this research will shed new light on the mechanisms of action of a crucial enzyme. In general, mechanistic/structural studies such as these, often offer a powerful means to the rational design of antimetabolites - perhaps anticancer agents.