MOLECULAR MECHANISMS OF PRPP-SYNTHETASE OVERACTIVITY

磷酸核糖焦磷酸合成酶 催化反应：核糖-5-磷酸+Mg 2 + PRPP + AMP。 通过这种酶形成PRPP代表了 嘌呤、嘧啶和吡啶的从头合成。 一 关于这种酶的大量信息 物理和动力学性质已经积累。 可是 关于编码PRPP的结构基因知之甚少- 合成酶或调节其表达。 由于PRPP是从头合成中的关键中间体， 嘌呤代谢的补救途径， 因为它是人体嘌呤合成的重要调节剂 体内平衡 PRPP-淀粉酶的超常活性 红细胞与X连锁综合征有关， 高尿酸血症，高尿酸尿症，早熟痛风， 肾结石 PRPP-S的几种突变形式具有 被部分定性。 动力学或调节的改变 属性（或两者的组合）似乎是负责 在这种突变体中观察到的超常活动。 的 这些催化活性变化的分子基础仍然存在 未知 因此，PRPP合成酶的遗传研究是相关的 提高对正常人类嘌呤代谢的理解， 以及人类疾病的发病机理。 这项建议的具体目标是： 表征编码正常人PRPP合成酶的cDNA; ii）产生抗人PRPP合成酶单特异性抗血清; iii） 表征细胞中的蛋白质和核酸异常 源自PRPP合成酶过度活性的受试者; iv） 从患有以下疾病的受试者中克隆突变形式的PRPP合成酶cDNA PRPP合成酶的超常变体。 私家侦探建议 从cDNA文库中原位分离PRPP合成酶cDNA 使用寡核苷酸探针进行杂交。 寡核苷酸将 衍生自获得的肽的部分氨基酸序列 纯酶。 将对重叠cDNA克隆进行测序 确定全长PRPP合成酶cDNA的结构 由此将推导出完整的氨基酸序列。 cDNA的同一性将通过比较 预测的氨基酸序列的肽， 寡核苷酸设计和杂交选择的基础。 然后将以这种方式获得的cDNA探针用于 表征正常和突变基因表达并克隆 来自淋巴细胞的突变型PRPP合成酶cDNA， 成纤维细胞来源于痛风患者， 酵素 从这些研究中，增强了对分子的理解， 痛风的病因是什么？ 此外，委员会认为， 表征一类潜在重要的突变， 即那些通过“超常”酶活性表达的 应该能让我们更深入地了解 蛋白质结构和异常催化功能。

Phosphoribosylpyrophosphate synthetase (PRPP-synthetase) catalyzes the reaction: Ribose-5-phosphate + Mg2+ PRPP + AMP. The formation of PRPP by this enzyme represents the first step in the de novo synthesis of purines, pyrimidines and pyridines. A considerable amount of information regarding this enzymes' physical and kinetic properties has been amassed. However, very little is known regarding the structural gene encoding PRPP- synthetase or the regulation of its expression. Since PRPP is a key intermediate in the de novo synthesis and salvage pathways of purine metabolism, the enzyme responsible for its synthesis is an important regulator of human purine homeostasis. Supernormal activity of PRPP-synthtase in erythrocytes is assoicated with an X-linked syndrome consisiting of hyperuricemia, hyperuricaciduria, precocious gout and nephrolithiasis. Several of these mutant forms of PRPP-S have been partially characterized. Alterations in kinetic or regulatory properties (or a combination of both) appear to be responsible for the supernormal activity observed in such mutants. The molecular bases for these alterations in catalytic activity remain unknown. Thus, genetic studies of PRPP-synthetase are relevant to improved understanding of normal human purine metabolism as well as the pathogenesis of human disease. The specific aims of this proposal are to: i) isolate and characterize the cDNA encoding normal human PRPP-synthetase; ii) raise monospecific antiserum to human PRPP-synthetase; iii) characterize protein and nucleic acid abnormalities in cells derived from subjects with PRPP-synthetase overactivity; iv) to clone mutant forms of PRPP-synthetase cDNA from subjects with supernormal variants of PRPP-synthetase. The P.I. proposes to isolate PRPP-synthetase cDNA from cDNA libraries by in situ hybridization using oligonucleotide probes. Oligonucleotides will be derived from partial amino acid sequence of peptides obtained from pure enzyme. Overlapping cDNA clones will be sequenced to determine the structure of full-length PRPP-synthetase cDNA from which the complete amino acid sequence will be deduced. The identity of the cDNA will be confirmed by comparison of predicted amino acid sequence to that of peptides which were not the basis for oligonucleotide design and by hybrid selection. cDNA probes obtained in this fashion will then be used to characterize normal and mutant gene expression and to clone mutant PRPP-synthetase cDNAs from lymphocytes and fibroblasts derived from gouty subjects with overactivity of the enzyme. From these studies, enhanced understanding of the molecular basis for this inherited form of gout should result. Furthermore, characterization of a potentially important class of mutations, i.e. those expressed through "supernomal" enzymatic activity should provide insights into the relationship between altered protein structure and abnormal catalytic function.