Structural Dissection of the HGPRT Reaction Mechanism

描述（申请人？摘要：近年来， 研究人员和他小组研究了次黄嘌呤-鸟嘌呤 磷酸核糖基转移酶（HGPRT）是弓形虫中一种关键的嘌呤补救酶 刚地。T.弓形虫是一种普遍存在的寄生原生动物， 的发病率和死亡率。长期研究目标是 设计HGPRT抑制剂作为治疗弓形虫病的药物。 首席调查员？在HGPRT上的前期工作提供了原子分辨率 结构信息、诱变数据和酶学结果， 一起解决几个关键问题，关于HGPRT反应机制。 这项工作提出了几个新的机械问题。另外的生物化学 并且需要结构信息来充分理解HGPRT作为一种酶。 这些新的知识将对我们理解 酶反应一般，它也将提供一个坚实的基础 合理设计靶向HGPRT的药物。 为了提供这些基本信息，主要研究者建议 （1）测试HGPRT反应机制的四个假设，关于 Leu 78-Lys 79活性位点肽键的顺反异构化 催化，活性位点环Il和IIT所起的作用，以及 T.（2）完成弓形虫HGPRT的测定 附加T.弓形虫HGPRT晶体结构，使用X射线衍射数据 已经掌握的组（野生型和具有多种配体的突变体）;（3） 解决了HGPRT与过渡态类似物结合的新结构和其他 现有化合物;和（4）确定HGPRT异四聚体或仅HGPRT异四聚体 T.弓形虫，并比较生化和 HGPRT同工酶的酶学性质。 三名合作者将协助主要研究者进行具体的、有限的 部分研究。大卫鲁什教授将检测我们的HGPRT同工酶 转化为T.弓形虫并将寄生虫裂解液返还给申请人？的实验室， 分析. Charles Grubmeyer教授将进行单周转动力学研究 野生型和突变T.弓形虫HGPRTs赫伯特教授将就 荧光研究。

DESCRIPTION (Applicant?s abstract): For the past few years the principal investigator and his group have studied hypoxanthine-guanine phosphoribosyltransferase (HGPRT), a key purine salvage enzyme in Toxoplasma gondii. T. gondii is a pervasive parasitic protozoan that is a leading cause of morbidity and mortality in AIDS patients. The long term research goal is to design HGPRT inhibitors that are useful as drugs to treat toxoplasmosis. The principal investigator?s prior work on HGPRT has provided atomic resolution structural information, mutagenesis data, and enzymological results that together address several key questions regarding the HGPRT reaction mechanism. This work has raised several new mechanistic questions. Additional biochemical and structural information is needed to fully understand HGPRT as an enzyme. This added knowledge will have fundamental impact on our understanding of enzyme reactions generally, and it will also provide a solid foundation for the rational design of drugs that target HGPRT. To provide this fundamental information, the principal investigator proposes to: (1) test four hypotheses on the HGPRT reaction mechanism, regarding cis-trans isomerization of the Leu78-Lys79 active site peptide bond during catalysis, the roles played by active site loops Il and IIT, and the basis for selective xanthine use by T. gondii HGPRT; (2) complete the determination of additional T. gondii HGPRT crystal structures, using x-ray diffraction data sets already in hand (wildtype and mutants with a variety of ligands); (3) solve new structures of HGPRT bound to transition state analogues and other existing compounds; and (4) determine whether HGPRT heterotetramers or only homotetramers exist in T. gondii, and to compare the biochemical and enzymological properties of the HGPRT isozymes. Three collaborators will assist the principal investigator in specific, limited portions of this research. Prof. David Roos will transfect our HGPRT isozymes into T. gondii and return parasite lysates to the applicant?s laboratory for analysis. Prof. Charles Grubmeyer will perform single-turnover kinetics studies of wildtype and mutant T. gondii HGPRTs. Prof. Herbert Cheung will consult on fluorescence studies.