PURINE METABOLISM IN TRICHOMONAS AND GIARDIA

我们目前研究计划的总体目标是建立一个模型， 来验证一种新方法的可行性， 对抗传染病 这种模式需要一个财团， 生物化学、药理学、分子生物学、结构 生物学、计算机图形学和有机合成的结合。 的 我们之前的代谢研究结果选择了胎儿三毛滴虫， 蓝氏贾第鞭毛虫作为两种寄生虫作进一步研究 由于它们在嘌呤和嘌呤的从头合成中的缺陷， 嘧啶核苷酸。 进一步的调查使我们的注意力集中在 T.胎儿次黄嘌呤-鸟嘌呤-黄嘌呤磷酸核糖基转移酶 （HGXPRTase）、T.胎儿IMP脱氢酶（IMPDH）和G.蓝氏鸟嘌呤 磷酸核糖基转移酶（GPRT酶），因为每种蛋白质被发现是 嘌呤补救中的关键酶;它们在每种情况下的抑制 抑制寄生虫的体外生长。 这些酶被纯化， 它们独特的底物特异性和催化性能， 初步研究表明。 编码这些酶的基因是 然后进行鉴定、克隆、测序和表达， 纯化的重组酶的天然形式。 他们分享 与它们的哺乳动物对应物相对低的序列同一性; T. 胎儿HGXPRTase，27.3%;兰伯氏菌GPRTase，低于20%; 胎儿T. IMPDH，34%。 T.胎儿HGXPRTase 分辨率为1.91。 这是一个异常紧凑的不对称 二聚体与许多独特的功能，在积极的口袋相比， 与人HGPRT酶的活性相同。 T.胎儿IMPDH为 分辨率也高达2.3A。 它主要是阿尔法贝塔 桶包装成两个扁平的C4对称四聚体。 虽然结构 的哺乳动物IMPDH是尚未可用于比较，我们的生化 关于寄生虫酶的数据已经表明， 霉酚酸，并确定了酶中的Cys 319作为催化剂， 亲核试剂 在下一个资助期内，我们打算：1） 通过定点突变， 诱变; 2）在稳态动力学分析中检查每种突变酶 改变的底物特异性和动力学常数; 3）分析 突变蛋白质的X射线晶体学结构及其相关 结构修改与功能变化; 4）使用计算机 图形DOCK程序筛选和识别潜在的抑制剂， 三种酶; 5）在酶测定中测试所选择的化合物， 在体外寄生虫细胞培养的铅，并修改的结构， 通过有机合成引导化合物。 的组合文库 还将在酶测定中筛选嘌呤类似物作为替代物 识别先导化合物的途径。 我们认为，坚实的基础 在此基础上取得重大成果， 近期

The overall goal of our present research proposal is to establish a model to verify the feasibility of a new approach to discover novel agents against infectious diseases. This model requires a consortium of researchers in biochemistry, pharmacology, molecular biology, structural biology, computer graphics and organic synthesis to work together. The results from our previous metabolic studies chose Tririchomonas foetus and Giardia lamblia as the two parasitic organisms for further investigations because of their deficiency in de novo synthesis of both purine and pyrimidine nucleotides. Further investigations focused our attention on T. foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), T. foetus IMP dehydrogenase (IMPDH) and G. lamblia guanine phosphoribosyltransferase (GPRTase), because each protein was found to be a pivotal enzyme in purine salvage; their inhibition in each case brought on arrested in vitro growth of the parasite. These enzymes were purified, and their distinctive substrate specificities and catalytic properties were demonstrated in preliminary studies. Genes encoding these enzymes were then identified, cloned, sequenced and expressed to yield large quantities of purified recombinant enzyme in their native forms. They share relatively low sequence identities with their mammalian counterparts; T. foetus HGXPRTase, 27.3 percent, G. lamblia GPRTase, lesser 20 percent; T.foetus IMPDH, 34 percent. The crystal structure of T. foetus HGXPRTase was identified at 1.91 resolution. It is an unusually compact asymmetric dimer with many distinctive features in the active pocket when compared with that of human HGPRTase. The crystal structure of T. foetus IMPDH was also determined up to 2.3A resolution. It is primarily an alpha beta barrel packed into two flat C4-symmetric tetramers. Though the structure of mammalian IMPDH is not yet available for comparison, our biochemical data on the parasite enzyme have already indicated an unusually high K1 for mycophenolic acid and identified Cys319 in the enzyme as the catalytic nucleophile. For the next granting period, we intend to: 1) generate a variety of mutants of the three enzyme proteins by site-directed mutagenesis; 2) examine each mutant enzyme in steady-state kinetic analysis for altered substrate specificities and kinetic constants; 3) analyze the structures of the mutant proteins in X-ray crystallography and correlate structural modifications with functional changes; 4) use the computer graphic DOCK program to screen and identify potential inhibitors of the three enzymes; 5) test the chosen chemical compounds in enzyme assays and in vitro parasite cell cultures for leads, and modify the structures of lead compounds through organic synthesis. Combinatorial libraries of purine analogs will be also screened in the enzyme assays as an alternative route to identify lead compounds. We believe that a solid basis has already been cast upon which significant results may be within reach in the near future.