REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

UMP合成酶是一种多功能蛋白质，含有两种口香酸 磷酸核糖转移酶(OPRTase)和核苷-5‘-单磷酸 脱羧酶(ODCase)。这两种酶是一系列 具有从头合成能力的所有生物体所需的六种酶 UMP是所有其他嘧啶核苷酸的前体。UMP合成酶 在快速生长的组织和细胞中升高，如肿瘤组织和细胞 起源，并一直是化疗的靶酶。基因 口腔性酸尿是一种疾病，已被描述，其直接原因是 缺失OPRTase和ODCase的UMP合成酶缺陷(I型)或仅缺失 ODCase(类型II)。慢性尿苷疗法克服了大部分 这种疾病的表现。我们已经提纯了人UMP合成酶以 用单抗亲和层析柱进行均一。一个人类的人民运动联盟 合成酶cDNA会被放入一个高产载体中，这样我们就可以 获得大量该蛋白用于结构/功能研究 它将包括X射线结晶学、机理研究和 定点突变。我们将识别氨基酸残基或 肽序列对于底物/抑制物结合是必不可少的，对于 酶催化，对蛋白质和蛋白质的构象状态 对活动的监管。与单一功能的比较研究 小鼠UMP合成酶的纯ODCase结构域--酵母ODCase 重组人UMP合成酶的OPRTase和ODCase结构域将允许 美国将评估中国是否有独一无二的特点 双功能蛋白。 我们的总体目标是确定双功能蛋白UMP 合成酶，简单地表示其共价连接的酶的总和 域，或者它是否也代表了一个具有附加动力学的实体 和调节特性，因为这种蛋白质(或可以 提供正常的产品，UMP)是人类正常生长所必需的 和发展。

UMP synthase is a multifunctional protein containing both orotate phosphoribosyltransferase (OPRTase) and orotidine-5'-monophosphate decarboxylase (ODCase). These two enzymes are the last of a sequence of six enzymes required by all organisms that have the de novo biosynthesis of UMP, a precursor of all other pyrimidine nucleotides. UMP synthase is elevated in rapidly growing tissues and cells such as those of tumor origin and has been a target enzyme for chemotherapy. The genetic disease, orotic aciduria, has been described and directly results from a defective UMP synthase lacking both OPRTase and ODCase (Type I) or only ODCase (Type II). Chronic uridine therapy overcomes most of the manifestations of the disease. We have purified human UMP synthase to homogeneity by a monoclonal antibody affinity column. A human UMP synthase cDNA will be placed in an overproducing vector so that we can obtain large quantities of this protein for structure/function studies which will include X-ray crystallography, mechanism studies, and site-specific mutagenesis. We will identify amino acid residues or peptide sequences are essential for substrate/inhibitor binding, for enzyme catalysis, for the conformational states of the protein and regulation of activity. Comparative studies with the monofunctional yeast ODCase, the pure ODCase domain of mouse UMP synthase and the recombinant OPRTase and ODCase domains of human UMP synthase will allow us to assess whether there are unique characteristics in the bifunctional protein. Our overall goal is to establish whether the bifunctional protein, UMP synthase, simply represents the sum of its covalently-joined enzyme domains or whether it also represents an entity with additional kinetic and regulatory characteristics, since this protein (or therapy that can provide the normal product, UMP) is necessary for normal human growth and development.