MECHANISTIC STUDIES OF DINUCLEAR METALLO-BETA-LACTAMASE

双核金属-β-内酰胺酶的机理研究

• 批准号: 6372903
• 负责人: WALTER L FAST
• 金额: $ 4.02万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6372903
• 关键词: active sites; bacterial proteins; beta lactamase; binding sites; biochemical evolution; chelating agents; drug resistance; enzyme inhibitors; enzyme mechanism; metalloenzyme; microorganism metabolism; site directed mutagenesis

The plasmid mediated spread of metallo-beta-lactamase poses a growing threat against the ability to combat antibiotic-resistant bacteria. This newly emerging enzyme has evolved to catalyze the hydrolysis of most beta- lactam antibiotics in clinical use. Understanding how this beta-lactamase works and how it can be inhibited is of significant economic and humanitarian interest. Found at the active site are two co-catalytic zinc ions, the functions of which are still poorly understood. This proposal employs site-directed mutagenesis and a zinc chelator to study metallo-beta- lactamases that can bind either one or two zinc ions selectively. Pre-steady- state and steady-state kinetics, mutagenesis spectroscopy, and inhibitor studies will be used to identify modes of inhibitor binding, the role of the protein environment and how the evolution of a second zinc binding site has resulted in a particularly effective and dangerous catalyst. Information about the catalytic mechanism and inhibitor binding will contribute to our understanding of dinuclear hydrolases and to the design of needed antibacterial agents combating the emerging threat of antibiotic-resistant bacteria.

质粒介导的金属β-内酰胺酶的扩散构成了对抗抗抗生素耐药细菌的能力的日益严重的威胁。这种新出现的酶已经发展为催化大多数β-乳用于临床用途的β-乳用于β-乳酰胺抗生素的水解。了解这种β-内酰胺酶的工作原理以及如何抑制它是具有重要的经济和人道主义利益。在活动位置发现的是两个共催化锌离子，其功能仍然很少了解。该提案采用位置定向的诱变和锌螯合剂来研究可以选择性地结合一种或两个锌离子的金属脱甲酰胺酶。稳态的状态和稳态动力学，诱变光谱和抑制剂研究将用于识别抑制剂结合模式，蛋白质环境的作用以及第二个锌结合位点的演化如何导致特别有效且危险的催化剂。有关催化机制和抑制剂结合的信息将有助于我们对二核水解酶的理解，以及设计需要打击抗生素耐药菌威胁的所需抗菌剂。