CLASS A BETA LACTAMASES

A 类 β 内酰胺酶

• 批准号: 2442519
• 负责人: Shahriar Mobashery
• 金额: $ 22.02万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2442519
• 关键词: X ray crystallography; acidity /alkalinity; beta lactamase; biochemical evolution; carboxypeptidase; cephalosporins; chemical kinetics; chemical synthesis; computer simulation; crystallization; drug resistance; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; hydrogen bond; mutant; nuclear magnetic resonance spectroscopy; penicillins; protease inhibitor; protein engineering; site directed mutagenesis

DESCRIPTION: The bacterial acquisition of beta-lactamases is the primary mechanism for their resistance to beta-lactam antibacterials (e.g., penicillins and cephalosporins). A multidisciplinary approach has been outlined for the study of the TEM-1 beta-lactamase, the prototypic member of the class A beta-lactamases. The proposed research benefits from the applicant's expertise in protein chemistry, organic synthesis, microbiology, and molecular biology. Crystallographic data and molecular modeling of penicillin and cephalosporin binding into the active site of the TEM-1 enzyme suggests the involvement of a hydrogen bond by the side chain of Asn-132. The role of Asn-132 residue in catalytic turnover will be probed by site-directed mutagenesis and specially designed substrates. The mechanistic function of Lys-73 which is highly conserved among class A beta-lactamases is unclear. Straight-forward analysis of mutants at this position would not provide a conclusive answer, so a novel NMR experiment is proposed to elucidate the function of this residue. Non-classical beta-lactam structures such as carbapenems are resistant to hydrolytic deactivation by beta-lactamases but the mechanistic basis is not understood. Molecular modeling has provided mechanistic insight into the process of turnover of carbapenems which may help. Based on these findings, new beta-lactamases molecules are proposed which are expected to be poor substrates for these enzymes, but are predicted to retain antibacterial property. Three new classes of mechanism-based inactivators have been proposed for beta-lactamases. These molecules will be synthesized and studied in detail as potential inactivators for both TEM-1 Q908R (class C) beta-lactamses. A novel cephalosporin has been designed which may be useful in delineating the structural basis for evolutionary kinship of penicillin-binding proteins (PBPs) and beta-lactamases.

描述：β-内酰胺酶的细菌获取是主要的 其对β-内酰胺抗菌抗菌的抗性机制（例如 青霉素和头孢菌素）。 多学科的方法已经 概述用于研究TEM-1β-内酰胺酶的研究， A类β-内酰胺酶。 拟议的研究受益于 申请人在蛋白质化学，有机合成，微生物学方面的专业知识， 和分子生物学。 晶体学数据和分子建模 青霉素和头孢菌素结合到TEM-1的活性位点 酶表明氢键与侧链的氢键参与 ASN-132。 ASN-132残基在催化周转中的作用将被探测 通过定点诱变和专门设计的底物。 这 LYS-73的机械功能，在A类中高度保守 β-内酰胺酶尚不清楚。 在此对突变体的直接分析 位置不会提供结论性的答案，因此新型的NMR实验是 提议阐明该残基的功能。 非古典 β-内酰胺结构（例如碳青霉）对水解具有抗性 β-内酰胺酶停用，但机械基础尚不清楚。 分子建模已提供了有关该过程的机械洞察力 碳青霉烯的营业额可能会有所帮助。 基于这些发现，新 提出了β-内酰胺酶分子，预计会很差 这些酶的底物，但预计将保留抗菌 财产。 基于机制的三类灭活因子已经 提议用于β-内酰胺酶。 这些分子将合成，并 详细研究了TEM-1 Q908R的潜在灭活因子（C类） β-内酰胺。 已经设计了一种新型的头孢菌素，可能很有用 在描述的结构基础上的进化亲属关系 青霉素结合蛋白（PBP）和β-内酰胺酶。