PROBING DRUG RESISTANCE IN B-LACTAMASE CRYSTALS BY RAMAN

但前提是。 这个项目的主要目标是阐明导致耐药的分子因素，8- 内酰胺酶家族。，8-1放线酶是由细菌产生的，它们破坏青霉素型 分子才能杀死它们的细菌目标。因此，8-1内酰胺酶本身就是抑制剂的靶标。 临床分离株表明，8-1内酰胺酶对其药物抑制物产生抗药性是通过 突变。这一建议将阐明SHV-1，8-1内酰胺酶与临床的化学反应 重要的药物，他唑巴坦、舒巴坦和克拉维酸；这些化合物起到了“自杀抑制剂”的作用。这个 反应将通过拉曼结晶学来表征-通过跟踪单晶中的反应 用拉曼显微镜观察酶的变化。药物被单独注射到含有晶体的母液中 对于β-内酰胺酶，抑制剂在不到一分钟的时间内完全扩散到晶体中，随后的反应在 通过拉曼差谱可以跟踪活性中心。使用适当形式的酶， 米氏络合物、酰基酶和最终产品的结构和种群可在 拉曼数据中的晶体。通过比较野生型酶的这些性质，以及具有 开发了一种抗药性抑制剂，独特地洞察了抗药性背后的分子机制 被获得了。8-1内酰胺酶的耐药形式是M691、M69L和M69V，选择的是它们的临床相关性。 拉曼方法表征单晶中中间体数量的能力将被用于 选择快速冷冻的最佳时间。然后，含有捕获的反应中间产物的晶体将被 以X射线结晶学为特征。 对于D类，8-1酰胺酶OXA-10和OXA-1，最近的研究表明，氨基甲酰化的赖氨酸发挥作用 在活性中心化学中起着关键作用。拉曼结晶学将被用来证实这一新的和有争议的 找到了。

PROVIDED. The principal goal of this project is to elucidate the molecular factors that lead to drug resistance in the ,8- lactamase family of enzymes. ,8-1actamases are produced by bacteria and they destroy penicillin-type molecules before they can kill their bacterial targets. Thus, ,8-1actamases themselves are targets for inhibitors. Clinical isolates demonstrate that ,8-1actamases develop resistance to their drug-inhibitors by undergoing point mutations. This proposal will elucidate the chemical reactions between a SHV-1 ,8-1actamase and the clinically important drugs, tazobactam, sulbactam and clavulanic acid; these compounds act as "suicide inhibitors". The reactions will be characterized by Raman crystallography - by following the reactions in single crystals of the enzyme using a Raman microscope. The drugs are injected separately into the mother liquor containing a crystal of p-lactamase, the inhibitors diffuse fully into the crystal in less than one minute and the subsequent reaction in the active site can be followed via the Raman difference spectrum. Using suitable forms of the enzyme, the structures and populations of the Michaelis complexes, acyl enzymes and final products can be defined in the crystals from the Raman data. By comparing these properties for the wild-type enzyme, and the enzyme that has developed a resistance to the inhibitors, unique insight into the molecular mechanisms underlying resistance will be gained. The resistant forms of the ,8-1actamase are M691, M69L and M69V, selected for their clinical relevance. The ability of the Raman method to characterize populations of intermediates in single crystals will be used to select optimal times for flash freezing. The crystals containing the trapped reaction intermediates will then be characterized by X-ray crystallography. For the class D ,8-1actamases OXA-10 and OXA-1 recent studies have indicated that a carbamylated lysine plays a key role in active site chemistry. Raman crystallography will be used to confirm this novel and controversial finding.