MECHANISTIC STUDIES OF DINUCLEAR METALLO-BETA-LACTAMASE

质粒介导的金属β-内酰胺酶的传播对对抗抗生素耐药细菌的能力构成了越来越大的威胁。这种新出现的酶已经进化成催化临床上使用的大多数β-内酰胺类抗生素的水解酶。了解这种β-内酰胺酶是如何工作的，以及如何抑制它，具有重大的经济和人道主义利益。在活性中心发现了两个共催化的锌离子，它们的功能仍然很少被了解。该方案利用定点突变和锌螯合剂来研究能选择性结合一个或两个锌离子的金属β-内酰胺酶。将使用稳态前和稳态动力学、诱变光谱和抑制剂研究来确定抑制剂结合的模式、蛋白质环境的作用以及第二个锌结合位点的进化如何导致特别有效和危险的催化剂。有关催化机制和抑制剂结合的信息将有助于我们理解双核水解酶，并有助于设计所需的抗菌剂来对抗抗生素耐药细菌的新威胁。

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.