STRUCTURAL STUDIES OF BACTERIAL METALLO-B-LACTAMASES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细菌对b-内酰胺类抗生素（青霉素和头孢菌素）的耐药性自从几年前首次出现在临床上以来，已经以惊人的速度发展。药物的抵消作用是通过水解N-C（=O）内酰胺键的胞外酶的表达来实现的。大约20种已知的β-内酰胺酶需要一个或两个当量的Zn（II）来获得活性。其中，大多数在两当量的Zn（II）下达到最大活性。然而，三种金属-b-内酰胺酶已被证明是完全活性的一个Zn（II）结合，这些被抑制的第二个Zn（II）的结合。已经从人类病原体中分离出几种金属-b-内酰胺酶，迄今为止还没有临床上可行的抑制剂。我们的目的是使用XAS补充磁共振实验，结合快速冷冻淬灭技术，表征稳定的静息状态和金属-b-内酰胺酶催化水解相关的催化中间体。短期目标是确定控制金属-b-内酰胺酶活性的结构特征。我们的长期重点是详细了解由第二金属离子决定的控制反应性的因素。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial resistance to the b-lactam family of antibiotics (penicillins and cephalosporins) has progressed at a tremendous rate since it first appeared clinically several years ago. Counteraction of the drugs is accomplished by the expression of extracellular enzymes which hydrolyze the N-C(=O) lactam bond. Approximately 20 of the known b-lactamases require one or two equivalents of Zn(II) for activity. Of these, the majority reach maximal activity with two equivalents of Zn(II). However, three metallo-b-lactamases have been shown to be fully active with one Zn(II) bound, and these are inhibited by binding of a second Zn(II). Several metallo-b-lactamases have been isolated from human pathogens, and to date there is no clinically viable inhibitor available. Our purpose is to use XAS to complement magnetic resonance experiments, in combination with rapid-freeze-quench techniques, to characterize stable resting states and catalytic intermediates relevant to metallo-b-lactamase catalyzed hydrolysis. The short-term goal is to determine the structural features that govern metallo-b-lactamase activity. Our long-term focus is a detailed understanding of the factors controlling reactivity that are determined by the second metal ion.