REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

UMP合成酶是一种多功能蛋白质， 磷酸核糖基转移酶和乳清酸核苷-5 '-单磷酸 脱羧酶（ODCase）。 这两种酶是最后一种 UMP从头生物合成所需的六种酶的序列， 所有其他嘧啶核苷酸的前体。 UMP合成酶是 在快速生长的组织和细胞中升高，例如 肿瘤起源，并已成为化疗的靶酶。 在 与肿瘤组织相比，遗传性疾病乳清酸尿症 并直接由缺陷的UMP合酶引起 缺乏OPRTase和ODCase（I型）或ODCase（I型 II）。 慢性尿苷治疗克服了 疾病 具有ODCase结构域的编码序列的cDNA 的UMP合酶，已被分离和表达的使用 合适的载体在酵母和E.缺乏这种基因的大肠杆菌突变体 蛋白 我们将继续尝试分离完整的cDNA， UMP合酶和/或位于5'端的OPRTase结构域的cDNA 来自pMEJ的ODCase结构域的编码序列。 的cDNA 将对UMP合酶的氨基酸序列进行测序， 待测蛋白质。 使用适当的表达方式 载体应该允许UMP合酶在合适的E. 大肠杆菌或酵母突变体。 UMP合成酶cDNA的编码区 将被改变。 这些新的cDNA将用于编程 OPRTase缺陷型和ODCase缺陷型E.大肠杆菌产生新的 截短的蛋白质，其仅补充一种缺陷。 这些 DNA将通过限制性酶切图谱和DNA测序进行分析， 确定整个氨基酸序列中 构成单独的OPRTase和ODCase催化结构域。 很少有直接的研究已经做了鉴定氨基酸侧 UMP合酶的两种酶活性所需的链。 使用酵母ODCase（其可以是 大量分离）也将启动此类研究 为了寻找使这种蛋白质结晶的必要条件 做X光检查 这些蛋白质研究应该有助于确定 UMP合酶的ODCase结构域中的位点，其中位点特异性 突变可以产生显著的改变。 当cDNA 对于UMP合酶变得可用，这样的研究可以扩展到 双功能蛋白和OPRTase结构域。

UMP synthase is a multifunctional protein containing both phosphoribosyltransferase (OPRTase) and orotidine-5'-monophosphate decarboxylase (ODCase). These two enzymes are the last of a sequence of six enzymes required for 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. In contrast to tumor tissue, the genetic disease, orotic aciduria, has been described and directly results from a defective UMP synthase lacking either both OPRTase and ODCase (Type I) or ODCase (Type II). Chronic uridine therapy overcomes the manifestations of the disease. A cDNA having the coding sequence for the ODCase domain of UMP synthase, has been isolated and expressed by use of appropriate vectors in yeast and E. coli mutants lacking this protein. We will continue to try to isolate the complete cDNA for UMP synthase and/or a cDNA for the OPRTase domain which lies 5' from the coding sequence of the ODCase domain of pMEJ. The cDNA for UMP synthase will be sequenced to allow the amino acid sequence of the protein to be determined. Use of appropriate expression vectors should allow expression of UMP synthase in appropriate E. coli or yeast mutants. The coding region of the UMP synthase cDNA will be altered. These novel cDNAs will be used to program OPRTase- deficient and ODCase deficient E. coli to produce new truncated proteins which complement only one deficiency. These DNAs will be analyzed by restriction mapping and DNA sequencing to determine which amino acids of the entire amino acid sequence constitute the individual OPRTase and ODCase catalytic domains. Few direct studies have been done to identify amino acid side chains required for both enzyme activities of UMP synthase. Protein modification studies using yeast ODCase (which can be isolated in large quantities) will initiate such studies as well as a search for conditions necessary to crystallize this protein for X-ray studies. These protein studies should aid in identifying sites in the ODCase domain of UMP synthase where site-specific mutations could produce significance modification. When the cDNA for UMP synthase becomes available such studies can be extended to the bifunctional protein and the OPRTase domain.