REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

UMP合成酶是一种多功能蛋白质，它既包含 磷酸核糖转移酶(OPRTase)和核苷-5‘-单磷酸 脱羧酶(ODCase)。这两种酶是一种 UMP从头合成所需的六种酶的序列， 所有其他的嘧啶核苷酸的前体。UMP合成酶是 在快速生长的组织和细胞中升高，如那些 肿瘤起源，一直是化疗的靶酶。在……里面 与肿瘤组织相比，遗传性疾病--口腔性酸尿症 被描述并直接由缺陷的UMP合成酶引起 缺少OPRTase和ODCase(类型I)或ODCase(类型 Ii)。慢性尿苷治疗克服了慢性尿毒症的表现 疾病。含有ODCase结构域编码序列的cDNA UMP合成酶的基因已被分离并通过使用 缺乏该基因的酵母和大肠杆菌突变体中的适当载体 蛋白。我们将继续努力分离完整的cDNAs UMP合成酶和/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。CDNA3 将对UMP合成酶进行测序，以允许氨基酸序列 待测蛋白质的含量。使用适当的表达方式 载体应允许UMP合成酶在适当的E. 大肠杆菌或酵母菌突变体。UMP合成酶基因的编码区 将会被改变。这些新颖的cDNA将被用来编程 OPRTase缺陷型和ODCase缺陷型大肠杆菌产生新的 仅补充一种缺陷的截短蛋白质。这些 DNA将通过限制图和DNA测序进行分析，以 确定整个氨基酸序列中的哪些氨基酸 构成各自的OPRTase和ODCase催化域。 很少有直接的研究来鉴定氨基酸的侧边。 UMP合成酶两种酶活性所需的链。 使用酵母ODCase的蛋白质修饰研究(可以 大量分离)也将启动这样的研究 为了寻找使这种蛋白质结晶的必要条件 用于X光检查。这些蛋白质研究应该有助于识别 UMP合成酶的ODCase结构域中的特定位点 突变可能会导致意义的改变。当cDNAs 随着UMP合成酶的出现，这种研究可以扩展到 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.