STRUCTURE, MECHANISM, AND INHIBITOR DESIGN FOR HGPRT

次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRTase)是主要的 人体嘌呤碱基抢救酶。在人类中，缺乏 HGPRTase导致以严重痛风为特征的Lesch-Nyhan综合征 以及一种鲜为人知的行为缺陷。在原生动物寄生虫中 酶负责嘌呤的利用，因为这些生物是 不能从头合成嘌呤，因此需要回收嘌呤 成长。原虫寄生虫恶性疟原虫引起疟疾， 据估计，每年造成200多万人死亡。长期目标 这项研究计划的目的是提供关于 人和恶性疟原虫HGPRTase允许的结构和机制 针对这些酶的物种特异性抑制剂的设计。两种酶 将通过在大肠杆菌中过度表达而产生。X射线晶体 人HGPRTase的结构已被测定为2.5埃 该项目的初步研究。将为以下方面建立结构 具有结合抑制物的人酶及其结构的初步研究 将尝试使用来自恶性疟原虫的酶。动力学和结合研究 将建立催化机理，底物和底物类似物 结合常数和抑制常数，以及限制整体 催化循环。动力学同位素效应将提供几何上的 由酶稳定的过渡态的结构。同位素 将用慢速底物测量IMP焦磷分解的效果， 亚膦酸根。化学溶剂解离的动力学同位素效应 5-P-核糖基-1-焦磷酸(PRPP)将与 在5-P-核糖转移酶反应中以PRPP为底物的酶。 这一比较将确定酶对稳定的贡献 酶的过渡态。过渡状态结构将是 用于设计过渡态抑制剂。晶体的比较 人和恶性疟原虫有无酶的结构 结合的抑制剂将提供有关机制的基本信息。 用于过渡态稳定。这两种转变的不同之处- 可以利用状态结构或酶结合位点来设计 特定物种的抑制剂。

Hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) is the major human enzyme of purine base salvage. In humans, the deficiency of HGPRTase causes Lesch-Nyhan syndrome which is characterized by severe gout and a poorly understood behavioral defect. In protozoan parasites the enzyme is responsible for purine utilization since these organisms are incapable of purine de novo synthesis and thus require purine salvage for growth. The protozoan parasite Plasmodium falciparum causes malaria which is estimated to cause over 2 million deaths per year. The long-term goal of this research program is to provide sufficient information on the structure and mechanisms of human and P. falciparum HGPRTases to permit the design of species-specific inhibitors for these enzymes. Both enzymes will be produced by overexpression in E. coli. The X-ray crystal structure of human HGPRTase has been determined to 2.5 Angstroms in preliminary studies for this project. Structures will be established for the human enzyme with bound inhibitors and the initial structure of the enzyme from P. falciparum will be attempted. Kinetic and binding studies will establish the catalytic mechanisms, substrate and substrate analogue binding and inhibition constants, and the steps which limit the overall catalytic cycle. Kinetic isotope effects will provide a geometric structures of the transition states stabilized by the enzymes. Isotope effects for IMP pyrophosphorolysis will be measured with a slow substrate, phosphonoacetate. Kinetic isotope effects for the chemical solvolysis of 5-P-ribosyl-1-pyrophosphate (PRPP) will be compared to those for the enzyme using PRPP as substrate in the 5-P-ribosyl transferase reaction. The comparison will establish the enzymatic contribution to stabilization of the enzymatic transition state. The transition state structure will be used to design transition state inhibitors. Comparison of crystal structures for the human and P. falciparum enzymes with and without inhibitors bound will provide fundamental information on the mechanisms for transition state stabilization. Differences in either transition- state structure or the enzymatic binding sites can be exploited to design species-specific inhibitors.