UMP SYNTHASE AND THE MOLECULAR BASIS OF OROTIC ACIDURIA

UMP合酶是一种双功能蛋白，催化最后两种酶， 尿苷5 ′-磷酸从头生物合成的步骤 （UMP）。 乳清酸磷酸核糖基转移酶 （OPRT）和乳清酸核苷-5 '-单磷酸脱羧酶（ODC），催化 乳清酸转化为乳清酸苷= 5 '-单磷酸（OMP） 以及OMP到UMP。 如从所述核苷酸序列确定的， UMP合成酶cDNA由480个氨基酸组成，具有一个 分子量52119 遗传性乳清酸尿症是一种常染色体隐性遗传病 与UMP的两种活动严重不足相关 合酶（I型），或仅缺乏ODC活性（I型 II）。 乳清酸尿症是唯一的特殊疾病的新生 嘧啶核苷酸的生物合成。 该项目的主要目标之一是确定 UMP合酶蛋白结构中的突变相关 乳清酸尿症 从大肠杆菌中克隆了UMP合酶cDNA序列， 乳清酸尿细胞将与正常序列进行比较。 在突变体中鉴定的序列改变将通过 各种方法，包括从 cDNA插入表达载体，等位基因杂交 特异性寡核苷酸或改变的限制性位点分析。 另一个主要目标是了解催化剂如何 UMP合成酶的结构域相互作用形成正常的双功能 蛋白 在原核生物和低等真核生物中， 存在于不同的蛋白质中。 在哺乳动物中， 催化结构域通过接头或接头序列连接。 通过改变接头的长度和氨基酸组成 肽，我们可以确定连接的结构要求 两个催化域。 通过使用表达载体， 也可以产生真核细胞， 具有OPRT和ODC活性或具有以下排列的单一蛋白质 的领域反转。 的稳定性和动力学性质 将改变的蛋白质与正常蛋白质进行比较， 双功能蛋白 乳清酸血症相关UMP合酶缺陷的分析 酸尿症将增加我们对遗传缺陷的理解， 它们对蛋白质结构和活性的影响。 的 表征催化域的相互作用将 提供有关酶进化和合作的信息， 核聚变和基底通道

UMP synthase is a bifunctional protein catalyzing the final two steps in the de novo biosynthesis of uridine 5' monophosphate (UMP). The two activities, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), catalyze the conversion of orotic acid to orotidine=5'-monophosphate (OMP) and OMP to UMP. As determined from the nucleotide sequence of the cDNA, UMP synthase is composed of 480 amino acids and has a molecular weight of 52,119. Hereditary orotic aciduria is a autosomal recessive disease associated with a severe deficiency of both activities of UMP synthase (Type I), or deficiency of only the ODC activity, (Type II). Orotic aciduria is the only specific disorder of de novo pyrimidine nucleotide biosynthesis described in humans. One of the primary objectives of this project is to identify the mutations in the structure of the UMP synthase protein associated with orotic aciduria. The sequence of the UMP synthase cDNA from orotic aciduria cells will be compared to the normal sequence. Sequence alterations identified in the mutant will be confirmed by various methods, including synthesis of the mutant protein from cDNA inserted into expression vectors, hybridization of allele specific oligonucleotides, or altered restriction site analysis. Another primary objective is to understand how the catalytic domains of UMP synthetase interact to form the normal bifunctional protein. In prokaryotes and lower eukaryotes, the two activities of UMP synthase reside in separate proteins. In mammals the two catalytic domains are joined by a linker or connector sequence. By changing the length and amino acid composition of the linker peptide we can determine the structural requirements for connecting the two catalytic domains. With the use of expression vectors we can also produce eukaryotic cells that synthesize separate proteins with OPRT and ODC activity or a single protein with the arrangement of the domains reversed. The stability and kinetic properties of the altered proteins will be compared to that of the normal bifunctional protein. Analysis of the defects in UMP synthase associated with orotic aciduria will increase our understanding of genetic defects and their effects on protein structure and activity. The characterization of the interaction of catalytic domains will provide information regarding enzyme evolution and cooperation, gone fusion, and substrate channeling.