METALLO BETA LACTAMASE FROM X MALTOPHILIA

分解β-内酰胺类和头孢菌素类抗生素的酶是 被称为β-内酰胺酶，细菌产生的β-内酰胺酶 细菌产生抗药性的常见方式。锌(II)- 含有β-内酰胺酶构成了一种不断增长和令人不安的 每摩尔含2摩尔锌(II)的一类β-内酰胺酶 酶，分解所有已知的青霉素和头孢菌素类抗生素， 不被克拉维酸抑制，也没有已知的抑制剂 活动。拟议研究的长期目标是 丙二醛不可逆缓蚀剂的合理设计与制备 含锌(II)的β-内酰胺酶。考虑到明显的结构性和 金属β-内酰胺酶的作用机制差异 目标只有在详细描述了一个 金属β-内酰胺酶每一不同亚类的成员。 然后，可以识别和利用这些酶之间的相似性 为了合理设计一种缓蚀剂。这项建议涉及 结构独特、具有重要生物医学意义的β-蛋白的研究 嗜麦芽窄食单胞菌内酰胺酶(L1)。这项建议的具体目标 主要研究内容有：(1)详细的金属结构表征 嗜麦芽窄食单胞菌的金属取代形式的结合部位 使用光谱、诱变和结合研究的内酰胺酶(2) 几种青霉素对该酶作用方式的阐明 和头孢菌素类抗生素使用稳态和预稳态 动力学研究，(3)总反应机理的确定 由LL和任何稳定的反应中间体使用数学 动力学速率分布和生化结果的模拟 研究，(4)将结构和力学成果整合到 确定酶的关键方面，可以作为靶点 开发基于反应或结构的新型抑制剂，以及(5) 检查化合物可能的抑制性能，在以下情况下 一旦被水解，就会产生活性的环外亚甲基基团。它是 希望这一集动力学、生化、模拟、 而光谱研究可以作为一种通用的策略用于 新型抗菌剂的制备和更好的抗炎方法 抗生素耐药性在细菌中的流行不断增加。

Enzymes that hydrolyze beta-lactams and cephalosporin antibiotics are called beta-lactamases, and their production by bacteria is the most common way that bacteria become antibiotic-resistant. The Zn(II)- containing beta-lactamases constitute an ever-growing and troubling class of beta-lactamases which contain 2 moles of Zn(II) per mole of enzyme, hydrolyze all known penicillin and cephalosporin antibiotics, are not inhibited by clavulanic acid, and have no known inhibitor of activity. The long-term objective of the proposed research is the rational design and preparation of irreversible inhibitors of the Zn(II)-containing beta-lactamases. Given the apparent structural and mechanistic differences among the metallo-beta-lactamases, this objective can only be realized after detailed characterization of a member from each distinct subclass of metallo-beta-lactamases. Similarities between the enzymes can then be identified and exploited for the rational design of an inhibitor. This proposal involves the study of the structurally-distinct, biomedically-important beta- lactamase (L1) from X maltophilia. The specific aims of this proposed research are: (1) detailed structural characterization of the metal binding sites of metal-substituted forms of the X. maltophilia beta- lactamase using spectroscopic, mutagenesis, and binding studies, (2) elucidation of the mode of action of the enzyme with several penicillin and cephalosporin antibiotics using steady-state and pre-steady state kinetics studies, (3) identification of the overall reaction mechanism used by Ll and of any stable reaction intermediates using mathematical simulations of kinetics rate profiles and results from biochemical studies, (4) integration of structural and mechanistic results to identify key aspects of the enzyme that can be targeted for the development of novel reaction- or structure-based inhibitors, and (5) examination of the possible inhibition properties of compounds, when once hydrolyzed produce reactive, exocyclic methylene groups. It is hoped that this novel integration of kinetics, biochemical, simulations, and spectroscopic studies can be used as a general strategy for the preparation of new antimicrobial agents and a better way to combat the ever-increasing prevalence of antibiotic resistance in bacteria.