PEPTIDE DEFORMAYLASE--MECHANISM AND INHIBITOR DESIGN

肽去甲酰基酶--机理和抑制剂设计

• 批准号: 6170308
• 负责人: Dehua Pei
• 金额: $ 24.14万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170308
• 关键词: X ray crystallography; active sites; aminopeptidase; antibacterial agents; chemical kinetics; combinatorial chemistry; drug design /synthesis /production; electron spin resonance spectroscopy; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate analog; metalloenzyme; peptide analog; prodrugs; site directed mutagenesis; sulfur aminoacid; ultraviolet spectrometry; zinc

The long-term objectives of this project are to understand the molecular mechanism of peptide deformylase, a small zinc-containing enzyme (169 amino acids in Escherichia coli), and design deformylase inhibitors and deformylase activated prodrugs as potential antibacterial agents. Ribosomal protein synthesis universally initiates with a methionine residue. In prokaryotes and the organelles of eukaryotes, the methionyl moiety attached to initiator tRNA is N-formylated prior to its incorporation. Following translational initiation, the N-formyl group is removed from the vast majority of bacterial proteins, a process catalyzed by the peptide deformylase. It has been demonstrated that proper deformylation of nascent polypeptides is essential for bacterial survival, as deletion of the deformylase gene (def) is lethal in E. coli. However, this deformylation process is apparently dispensable in eukaryotes. Therefore, deformylase appears to be an ideal target for developing broad-spectrum antibiotics, since formylation is a conserved feature in all eubacteria. Specific aims include: 1) Determination of the substrate specificity of deformylase using synthetic substrate analogues and combinatorial library screening; 2) Determination of catalytic mechanism using isotope exchange and chemical trapping experiments; 3) Determination of its 3-D structure by X-ray crystallography; 4) Determination of the function of its Zn2+ ion and identification of active-site residues by spectroscopic methods and site-directed mutagenesis; and 5) Design and synthesis of deformylase inhibitors and prodrugs that could be activated by deformylase action in vivo.

该项目的长期目标是了解分子 肽去甲酰基酶的机制，一种小型含锌酶（169） 大肠杆菌中的氨基酸），并设计去甲酰化酶抑制剂和 去甲酰酶激活的前药作为潜在的抗菌剂。 核糖体蛋白质合成普遍以甲硫氨酸起始 残留物。在原核生物和真核生物的细胞器中，甲硫氨酰 与起始子 tRNA 相连的部分在其被 N-甲酰化之前 合并。翻译起始后，N-甲酰基是 从绝大多数细菌蛋白质中去除的过程 由肽去甲酰酶催化。已经证明 新生多肽的适当去甲酰化对于细菌的生长至关重要 生存，因为去甲酰化酶基因 (def) 的缺失在大肠杆菌中是致命的。 大肠杆菌。然而，这种去甲酰化过程显然是可有可无的 真核生物。因此，去甲酰酶似乎是一个理想的靶标。 开发广谱抗生素，因为甲酰化是一种保守的 存在于所有真细菌中。具体目标包括： 1) 确定 使用合成底物的去酰基酶的底物特异性 类似物和组合文库筛选； 2) 测定 使用同位素交换和化学捕获的催化机制 实验； 3) X射线测定其3-D结构 晶体学； 4) 其Zn2+离子功能的测定和 通过光谱方法鉴定活性位点残基 定点诱变； 5)去甲酰酶的设计与合成 可以通过去甲酰酶作用激活的抑制剂和前药 体内。