GLUTAMINE AMIDOTRANSFERASE STRUCTURE/FUNCTION/REGULATION

本研究的目的是研究（一）组织 和调节编码谷氨酰胺转移酶的基因 酶，（ii）结构与谷氨酰胺酰胺的关系 转移功能和酶催化机制， 两个不同的谷氨酰胺酰胺转移结构域，和（iii）所述 （4Fe-4S）中心在酰胺转移酶和 第二种是特性很好的酶，乌头酸酶。 实验 方法将强调分子生物学。 克隆的基因 使用的包括：细菌trpEG（邻氨基苯甲酸合酶）、E.杆菌 pyrG（CTP合成酶）、E. coli和B. 枯草杆菌紫 （酰胺磷酸核糖基转移酶），和一个大簇的pur基因 来自B。 枯草杆菌。 此外，猪心顺乌头酸酶cDNA将被 克隆的 具体目标是：（i）采用序列比较 同源谷氨酰胺酰胺转移结构域， 保守的，可能必需的氨基酸残基，定点的 诱变以取代推断的必需残基， 酶化学来确定功能残基的作用 在催化方面。 同样的方法将用于调查 三种酰胺转移酶的变构调节位点。(ii)的 至少12个基因簇的克隆和核苷酸序列 参与B中嘌呤核苷酸的合成。枯草杆菌将是 完成 这一簇可能包含了所有的基因， 新途径IMP的表达和独特的调节。 基因簇的研究。 (iii)体外和体内突变 分析将用于研究启动子和调控区 大肠 coli purF. 一个计划是描述隔离一个未链接的 调控突变，然后将用于克隆推定的 反式作用调节基因 (iv)B. 枯草 氨基磷酸核糖基转移酶，涉及NH 2-末端 十一肽剪切和（4Fe-4S）中心的组装，将是 在细菌和CHO细胞中研究。 定点突变是 计划确定十一肽前导是否在 在装配的（4Fe-4S）中心，以及是否加工 领导者需要因素或自我催化。 这个B。 枯草 酶是一个很好的模型，目前无法获得的人类 这种酶可能在痛风中起作用。(v)克隆及序列 顺乌头酸酶cDNA的分析将启动对第二个（4Fe- 4S）酶，并将补充X射线结构分析。 抑制肿瘤细胞生长的药物，如阿西霉素， 谷氨酰胺酰胺转移酶的特异性抑制剂，说明了 这组酶在细胞生长中的重要作用， 这些酶的重要性。

The objectives of this research are to study the (i) organization and regulation of genes encoding glutamine amidotransferase enzymes, (ii) relationships of structure to glutamine amide transfer function and mechanisms for catalysis in enzymes having two different glutamine amide transfer domains, and (iii) the role(s) of the (4Fe-4S) centers in an amidotransferase and a second, well characterized enzyme, aconitase. The experimental approach will emphasize molecular biology. The cloned genes to be used include: bacterial trpEG (anthranilate synthase), E. coli pyrG (CTP synthetase), E. coli and B. subtilis purF (amidophosphoribosyltransferase), and a large cluster of pur genes from B. subtilis. In addition, pig heart aconitase cDNA will be cloned. The specific aims are: (i) Employ sequence comparisons of homologous glutamine amide transfer domains to infer conserved, possibly essential amino acid residues, sitedirected mutagenesis to replace inferred essential residues, and techniqus of enzyme chemistry to determine the role of functional residues in catalysis. This same approach will be used to investigate allosteric regulatory sites in three amidotransferases. (ii) The cloning and nucleotide sequence of a cluster of at least 12 genes involved in purine nucleotide synthesis in B. subtilis will be completed. This cluster likely contains all of the genes for the de novo pathway to IMP. The expression and unique regulation of the gene cluster will be studied. (iii) In vitro and in vivo mutational analyses will be used to study the promoter and regulatory region of E. coli purF. A plan is described to isolate an unlinked regulatory mutation which will then be used to clone the putative trans-acting regulatory gene. (iv) The maturation of B. subtilis amidophosphoribosyltransferase, which involves NH2-terminal undecapeptide clipping and assembly of a (4Fe-4S) center, will be studied in bacterial and CHO cells. Site-directed mutations are planned to determine whether the undecapeptide leader has a role in the assembly of the (4Fe-4S) center, and whether processing of the leader requires factors or is autocatalytic. This B. subtilis enzyme is a good model for the presently unavailable human enzyme, which may have a role in gout. (v) Cloning and sequence analysis of aconitase cDNA will initiate studies on a second (4Fe- 4S) enzyme and will complement the X-ray structural analysis. Inhibition of tumor cell growth by drugs such as acivicin, a specific inhibitor of glutamine amidotransferases, illustrates the essential role of this group of enzymes in cell growth and the importance of these enzymes.