STRUCTURE & DYNAMICS OF METALLO BETA LACTAMASE

项目2涉及结构的阐明和 来自临床重要厌氧菌的金属-β-内酰胺酶的动力学 有机体，脆弱拟杆菌。该项目的目标有两个： 获取抗生素关键成分分子的信息 生物体的抵抗力，并利用该系统作为 研究多肽链动力学和 酶的功能。由于 NMR 是获取位点的主要方法 溶液中多肽动力学的具体信息，重点 该项目是对 B. fragilis metallo-beta- 的广泛核磁共振研究 内酰胺酶，一种不含野生型酶且与抑制剂复合的酶 根据动力学专门设计的突变蛋白， 野生型蛋白质的结构和动态信息。 将对主动形式进行完整的共振分配 酶，含有两个锌离子。初步数据非常好，并且 表明可以使用以下组合进行此类分配 基于 Calpha 和 CO 的策略。完成共振作业也将 制备酶与底物类似物的复合物，以及 将确定该复合物的溶液结构。 将通过 NMR 研究游离酶的多肽动力学 配合物，使用 15N、13C 和 2H 弛豫测量。该研究 将为金属-β-内酰胺酶的诱变提供基础 （项目 3）和阐明反应途径（项目 4）。 第四个具体目标涉及局部结构的研究 酶的活性位点，使用镉取代的金属-β- 内酰胺酶。配体和溶剂的身份和键合 金属部位的暴露量可以从这些测量中获得， 以及金属离子解离常数的估计。 最终的具体目标将取决于各方之间的相互作用 计划项目的组成部分。共振作业和放松 将对根据累积的突变体设计进行测量 野生型动力学信息（项目 2）、动力学研究（项目 3）和计算（项目4）。结构将被确定为 通过 NMR 或 X 射线晶体学在溶液中选择突变体 合作者奥斯纳特·赫茨伯格博士。获得的信息来自 计划项目各组成部分之间的协同互动 应该为酶动力学的作用提供重要的见解 行动。

Project 2 is concerned with the elucidation of the structure and dynamics of a metallo-beta-lactamase from a clinically important anaerobic organism, Bacteroides fragilis. The goals of the project are two-fold, to obtain information on a molecule that is a key component of antibiotic resistance in the organism, and to utilize this system as one of the paradigms to investigate coupling between polypeptide chain dynamics and enzymatic function. Since NMR is the premier method for obtaining site- specific information on polypeptide dynamics in solution, the focus of the project is a extensive NMR study of the B. fragilis metallo-beta- lactamase, the wild type enzyme free and in complex with an inhibitor and mutant proteins specifically designed on the basis of the kinetic, structural and dynamic information available on the wild-type protein. Complete resonance assignments will be made for the active form of the enzyme, which contains two zinc ions. Preliminary data are excellent, and indicate that such assignments will be possible using a combination of Calpha and CO-based strategies. Complete resonance assignments will also be made for a complex of the enzyme with a substrate-analog, and a solution structure determination will be made for this complex. Polypeptide dynamics will be investigated by NMR for both the free enzyme an the complex, using 15N, 13C and 2H relaxation measurements. The study will provide the basis for mutagenesis of the metallo-beta-lactamase (Project 3) and for the elucidation of the reaction pathway (Project 4). The fourth specific aim concerns the study of local structure in the active site of the enzyme, using cadmium-substituted metallo-beta- lactamase. The identity and bonding of the ligands and the solvent exposure of the metal site can be obtained from these measurements, together with an estimate of the dissociation constants of the metal ions. The final specific aim will follow from the interactions between the components of the Program Project. Resonance assignments and relaxation measurements will be made for mutants designed on the basis of accumulated information form wild-type dynamics (Project 2), kinetic studies (Project 3) and calculations (Project 4). Structures will be determined for selected mutants either in solution by NMR or by X-ray crystallography by a collaborator, Dr. Osnat Herzberg. Information obtained from the synergistic interaction between the components of the Program Project should provide important insights into the role of dynamics in enzyme action.