NMR SOLUTION STRUCTURE OF THE MMOB COMPONENT

MMOB 组件的 NMR 解决方案结构

基本信息

批准号：
2521187
负责人：
George T. Gassner
金额：
$ 3.02万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-06-28 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/2521187
关键词：
active sites calorimetry catalyst chemical kinetics enzyme complex enzyme mechanism enzyme structure ionic bond methane monooxygenase nuclear magnetic resonance spectroscopy oxygenases protein structure site directed mutagenesis

项目摘要

The soluble methane menooxygenase system isolated from M. capsulatus (Bath) consists of a reductase (MMOR), coupling protein (MMOB), and a hydroxylase (MMOH). The 16.5 kDa MMOB component has a profound influence on the type of chemistry catalyzed at the active site of MMOH. In a still unknown mechanism, MMOB directs the evolution of a series of oxygen intermediates that lead to the insertion of an atom of molecular oxygen into a CH bond of methane. In the absence of MMOB, MMOH still accepts electrons from substrate specificity of MMOH. This broad substrate specificity makes MMOH valuable system in both aqueous phase organic synthesis, and in the detoxification of contaminated environmental sites. High resolution homonuclear and heteronuclear NMR experiments will be used to solve the solution structure of MMOB. The three dimensional NMR structure will provide greater insight into the way in which MMOB tunes the reactivity potentials of the MMOH iron center. Searches based on the complementarity of the topologies and electrostatic surface potentials of MMOH and MMOB will be used in conjunction with chemical cross-linking data to identify probable MMOB binding sites on the surface of MMOH. Proposed binding sites will be confirmed through titration calorimetric studies involving synthetic peptides representative of secondary structural motifs in interface region. Individual amino acid side chains involved in the complex formation will be identified in high resolution NMR studies of complexes of MMOB with synthetic peptides. Amino acid side chains found to be involved in the binding interaction will be sequentially mutated to alanine. The relative contribution of individual amino acid side chains the macroscopic stability of the complexes formed between MMOB and MMOH will be measured based on calorimetric measurements.

从 M. capsulatus 中分离出的可溶性甲烷双加氧酶系统（Bath）由还原酶（MMOR）、偶联蛋白（MMOB）和羟化酶（MMOH）。 16.5 kDa MMOB成分影响深远 MMOH 活性位点催化的化学类型。在静止的机制未知，MMOB指导一系列氧的演化导致分子氧原子插入的中间体成甲烷的CH键。在没有MMOB的情况下，MMOH仍然接受来自 MMOH 底物特异性的电子。这广阔的底材 MMOH 的专一性使其在水相和有机相中都很有价值合成以及污染环境场所的解毒。将使用高分辨率同核和异核核磁共振实验求解MMOB的解结构。三维核磁共振结构将提供对 MMOB 调优方式的更深入了解 MMOH 铁中心的反应电位。搜索基于拓扑和静电表面电势的互补性 MMOH和MMOB将与化学交联数据结合使用识别 MMOH 表面上可能的 MMOB 结合位点。建议的结合位点将通过滴定量热研究来确认涉及代表二级结构基序的合成肽在界面区域。参与的单个氨基酸侧链复杂的形成将在高分辨率核磁共振研究中被识别 MMOB 与合成肽的复合物。发现氨基酸侧链参与结合相互作用的将依次突变为丙氨酸。各个氨基酸侧链的相对贡献 MMOB 和 MMOH 形成的复合物的宏观稳定性将根据量热测量进行测量。