Structural Dissection of the HGPRT Reaction Mechanism

描述(申请人？S摘要)：过去几年的校长 研究人员和他的团队研究了次黄嘌呤-鸟嘌呤 弓形虫中关键的嘌呤抢救酶--磷酸核糖基转移酶 贡地。弓形虫是一种普遍存在的寄生原虫，是导致弓形虫感染的主要原因。 艾滋病患者的发病率和死亡率。长期的研究目标是 设计HGPRT抑制剂，作为治疗弓形虫病的药物。 首席研究员S之前在HGPRT上的工作提供了原子分辨率 结构信息、诱变数据和酶学结果 共同解决关于HGPRT反应机制的几个关键问题。 这项工作提出了几个新的机械问题。附加生化 而结构信息是完全理解HGPRT作为一种酶所必需的。 这一增加的知识将对我们对 一般的酶反应，这也将为 合理设计针对HGPRT的药物。 为了提供这一基本信息，首席调查员建议 TO：(1)检验关于HGPRT反应机理的四个假设，涉及 Leu78-Lys79活性部位肽键的顺反异构化 催化，活性中心环Il和Iit所起的作用，以及 弓形虫HGPRT选择性利用黄嘌呤；(2)完成黄嘌呤的测定 额外的弓形虫HGPRT晶体结构，使用x射线衍射数据 已有套装(野生型和具有各种配体的突变体)；(3) 解决与过渡态类似物结合的HGPRT的新结构 现有化合物；以及(4)确定HGPRT是异构体还是仅 在弓形虫中存在同源异构体，并比较生化和 HGPRT同工酶的酶学性质。 三名合作者将协助首席调查员在特定的、有限的 这项研究的部分内容。David Roos教授将把我们的HGPRT同工酶 进入弓形虫并将寄生虫裂解物退还给申请人？S实验室 分析。查尔斯·格鲁梅耶教授将进行单周转动力学研究 野生型和突变型弓形虫HGPRTS。张学良教授将就以下事项提供意见 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.