MOLECULAR MECHANISMS OF PRPP-SYNTHETASE OVERACTIVITY

PRPP 合成酶过度活性的分子机制

• 批准号: 3462888
• 负责人: THOMAS D PALELLA
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3462888
• 关键词: complementary DNA; enzyme mechanism; enzyme structure; fibroblasts; gene expression; genetic library; gout; human subject; human tissue; in situ hybridization; laboratory rabbit; lymphocyte; molecular cloning; molecular genetics; monoclonal antibody; mutant; nucleic acid hybridization; oligonucleotides; protein sequence; structural genes

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.

磷酸核糖焦磷酸合成酶(PRPP-合成酶) 催化反应：核糖-5-磷酸+镁+PRPP+AMP。 这种酶形成的PRPP代表了 嘌呤、嘧啶和吡啶的从头合成。一个 关于这种酶的大量信息 物理和动力学性质已经积累起来。然而，非常 对编码PRPP的结构基因知之甚少。 合成酶或其表达的调节。 由于PRPP是从头合成的关键中间体， 嘌呤代谢的挽救途径，负责的酶 因为它的合成是人类嘌呤的重要调节因子 动态平衡。中国人PRPP合酶活性超常 红细胞与一种X连锁综合征相关 高尿酸血症、高尿酸尿症、性早熟痛风和 肾结石。PRPP-S的这些突变形式中的几个 已经被部分定性了。动力或监管方面的变化 属性(或两者的组合)似乎要对此负责 在这种突变体中观察到的超常活动。这个 这些催化活性变化的分子基础仍然存在 未知。因此，对PRPP合成酶的遗传学研究是相关的。 为了更好地理解人类正常的嘌呤代谢， 以及人类疾病的发病机制。 这项提议的具体目的是：i)隔离和 对编码正常人PRPP合成酶的基因进行鉴定； Ii)制备抗人PRPP合成酶的单特异性抗血清；iii) 细胞中蛋白质和核酸异常的特征 源自具有PRPP合成酶过度活性的受试者；iv)至 慢性阻塞性肺疾病患者PRPP合成酶基因突变形式的克隆 PRPP合成酶的超常变异。私家侦探建议 用原位杂交技术从文库中分离PRPP合成酶基因 使用寡核苷酸探针进行杂交。寡核苷酸将 由获得的多肽的部分氨基酸序列衍生而来 从纯酶中提取。重叠的cdna克隆将被测序 测定全长PRPP-合成酶基因的结构 由此可以推断出完整的氨基酸序列。 通过比较，确定该基因的同源性。 预测的氨基酸序列与未预测的多肽的氨基酸序列 这是寡核苷酸设计和杂交选择的基础。 以这种方式获得的cdna探针随后将用于 鉴定正常和突变基因的表达并进行克隆 突变型PRPP合成酶基因从淋巴细胞和 痛风患者的成纤维细胞具有过度的 酵素。 从这些研究中，加深了对分子的理解 这种遗传性痛风的基础应该是结果。此外， 一类潜在的重要突变的特征， 即通过“超常”酶活性表达的那些 应该提供对改变的关系的见解 蛋白质结构和催化功能异常。