GLUTAMINE AMIDOTRANSFERASE STRUCTURE/FUNCTION/REGULATION

本研究的目的是研究(一)组织 和编码谷氨酰胺氨基转移酶基因的调控 酶，(II)结构与谷氨酰胺的关系 酶的传递函数和催化机理 两个不同的谷氨酰胺转移域，以及(Iii) (4Fe-4S)中心在氨基转移酶和氨基转移酶中的作用(S) 第二，特性良好的酶，乌头酸酶。实验性的 方法将强调分子生物学。克隆的基因 可使用的包括：细菌trpEG(邻氨基苯甲酸合成酶)，大肠杆菌 PYRG(CTP合成酶)、大肠杆菌和枯草芽孢杆菌 (氨基磷酸核糖基转移酶)和一大簇pur基因 来自枯草杆菌。此外，猪心乌头酸酶基因将被 克隆的。具体目标是：(I)使用序列比较 同源谷氨酰胺转移结构域的关系 保守的，可能是必需的氨基酸残基，定点定向 用于取代推断的必需残基的诱变和技术 酶化学来确定功能残基的作用 在催化作用下。同样的方法将被用来调查 三种酰胺转移酶的变构调节位点。(Ii) 至少12个基因簇的克隆和核苷酸序列分析 在枯草杆菌中参与嘌呤核苷酸合成的将是 完成。这一簇很可能包含De的所有基因 通向IMP的Novo途径。基因的表达和独特的调控 将对基因簇进行研究。(三)体外和体内突变 分析将用于研究启动子和调控区域 E.ColiPurF.描述了一项计划，以隔离未链接的 调节性突变，然后将用于克隆推定的 反式作用调控基因。(Iv)枯草杆菌的成熟 氨基磷酸核糖基转移酶，涉及氨基末端 十一肽的剪裁和组装一个(4Fe-4S)中心，将 在细菌和CHO细胞中进行了研究。定点突变是 计划确定十一肽领导人是否有作用 在(4Fe-4S)中心的组装中，是否加工 领导者需要因子，或者是自动催化的。这株枯草芽孢杆菌 对于目前无法获得的人类来说，酶是一个很好的模型 酶，这可能在痛风中起作用。(V)克隆和测序 对乌头酸酶的分析将启动对第二个(4Fe-)的研究 4S)酶，将补充X-射线结构分析。 阿昔维星等药物对肿瘤细胞生长的抑制作用 谷氨酰胺氨基转移酶的特异性抑制剂，说明了 这组酶在细胞生长和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.