INORGANIC POLYPHOSPHATES IN METABOLISM

代谢中的无机多磷酸盐

• 批准号: 3277239
• 负责人: NELSON F B PHILLIPS
• 金额: $ 18.48万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277239
• 关键词: Bacillus stearothermophilus; Mycobacterium tuberculosis; Propionibacteriaceae; biochemical evolution; chemical binding; chemical chain length; electron microscopy; enzyme mechanism; enzyme structure; enzyme substrate; gel electrophoresis; genetic library; glucokinase; high energy compound; human tissue; microorganism metabolism; phosphorylase kinase; phosphotransferases; polyphosphates; protein sequence; radionuclides; radiotracer; tissue /cell culture; yeasts

It is hypothesized that inorganic polyphosphate (poly(P)) served as a primitive source of energy and during the course of evolution, there may have been a transition from poly(P) to ATP. The overall objective of this proposal is to characterize enzymes involved in poly(P) metabolism so as to understand the specific mechanistic and molecular structures that result in the utilization of the high energy phosphoryl bond of poly(Ps). The information obtained will then be applied to the ultimate objective of elucidating the role of poly(P) in vivo. The proposed work will be focused on two such enzymes, polyphosphate/ATP glucokinase (poly(P)/ATP GK) and endopolyphosphatases/Exopolyphosphatases (endo PP/exo PP). the poly(P)/ATP GK utilizes either poly(P) of ATP and both activities are catalyzed by the same protein. Specific aims of experiments include (a) the isolation and sequencing of peptides at the ATP and poly(P) binding and/or catalytic sites of the enzyme from Propionibacterium shermanii, mycobacterium tuberculosis, and Nocardia minima; (b) determination of the kinetic and catalytic mechanisms and (c) isolation and sequencing of the poly(P)/ATP GK gene of P. shermanii and M. tuberculosis from available genomic libraries. The amino acid sequence of the ATP site will be determined using affinity analogs of ATP like 8-Azido-ATP and the lysine specific reagent, sulfonsuccinimidyl- hydroxyphenyl propionate. The purpose will be to compare these with known ATP sequences of glucokinase and hexokinases from higher life forms so as to determine if there are similarities in the ATP sites which will indicate an evolutionary continuity from ancient to more recent evolutionary forms. The poly(P) site peptides will be isolated using phosphate binding-site direct reagents like azidonitrophenyl pyrophosphate and pyridoxal phosphate. The organisms in the proposed studies are classified in the order Actinomycetales and are considered to be primitive. The poly(P) activity is greater than that of ATP in the older members (e.g., P. shermanii) while in the more recent Nocardia, it is reversed. Comparative structural and mechanistic information on the poly(P) and ATP sites may provide information on how substrate binding sites evolve. Protein sequence deduced from the DNA sequence will enable the comparison of whole protein structures with other glucokinases. Isolation of the poly(P)/ATP GK gene will be from the available lambdagt11 and lambdaEMBL3 genomic libraries. the second major focus will be on the characterization of the endo PP and exo PP to determine (a) if the two activities are the catalytic properties of a single protein; (b) whether different endo PP are present that are specific for certain sizes of poly(P). The final objective will be to demonstrate and identify poly(P) synthesizing enzyme(s) in animal cells. the presence of such enzymes in animal cells will be the first direct demonstration of the involvement of poly(P) in in vivo metabolic functions of mammalian cells.

假设无机多磷酸盐（poly（p））用作 原始的能源来源以及在进化过程中，可能 已经是从聚（P）到ATP的过渡。 总体目标 该建议是为了表征参与聚（P）代谢的酶 以了解特定的机械和分子结构 这导致利用高能磷酸键 聚（PS）。 然后获得的信息将应用于最终 阐明聚（P）在体内作用的目的。 拟议的工作 将集中于两种这样的酶，聚磷酸/ATP葡萄糖激酶 （poly（p）/ATP GK）和内多磷酸酶/外磷酸酶（内托酶 PP/EXO PP）。 聚（P）/ATP GK使用ATP的Poly（P）和两者都 活性被相同的蛋白质催化。 具体目标 实验包括（a）肽在肽处的分离和测序 酶的ATP和聚（P）结合和/或催化位点 谢尔马尼丙菌病，结核分枝杆菌和诺卡氏菌 minima; （b）测定动力学和催化机制以及（c） shermanii和 M.来自可用基因组文库的结核病。 氨基酸 ATP位点的序列将使用ATP的亲和力类似物确定 像8-氮杂 - ATP和赖氨酸特异性试剂一样 丙酸羟基苯基。 目的是将它们与 来自较高寿命形式的葡萄糖激酶和己糖苷酶的已知ATP序列 以确定ATP站点中是否有相似之处 表明从古代到最近的进化连续性 进化形式。 将使用poly（p）位点肽使用 磷酸盐结合位置直接试剂，例如叠氮硝基苯基 焦磷酸盐和吡啶还毒磷酸盐。 提议中的生物 研究按顺序进行放线菌进行分类，并被认为 是原始的。 聚（P）活性大于ATP的活性 年长的成员（例如P. Shermanii）在最近的Nocardia中 相反。 比较结构和机械信息 聚（P）和ATP位点可以提供有关底物结合的信息 站点的发展。 从DNA序列推导的蛋白质序列将启用 整个蛋白质结构与其他葡萄糖激酶的比较。 聚（P）/ATP GK基因的分离将来自可用的 lambdagt11和lambdaembl3基因组库。 第二个主要重点 将是关于Endo PP和Exo PP的表征 （a）如果这两个活动是单个活动的催化特性 蛋白质; （b）是否存在特定于 某些尺寸的聚（P）。 最终目标是展示和 识别动物细胞中的聚（P）合成酶。 存在 动物细胞中的这种酶将是第一个直接示范 聚（P）参与哺乳动物的体内代谢功能 细胞。