ISOTOPIC PROBES OF ENZYMATIC REACTION MECHANISMS

本文件中描述的研究的广泛、长期目标 建议的目的是阐明复杂的关系 生物系统中结构和功能之间的关系。主要关注点 本申请的目的是针对催化和 细菌磷酸三酯酶的结构。这种酶催化 通过水解酶解毒或有机磷神经毒素 磷-氧和磷-氟键。磷酸三酯酶 在扩散时催化最佳底物的水解- 控制极限和二价阳离子是催化所必需的 活动。主要金属离子的作用将通过以下方式进行探讨 用多种光谱取代天然锌离子 活性阳离子(CD2、Mn2、Ni2、CO2和Cu2)。配基 金属中心的环境将用~(113)Cd-核磁共振进行研究 光谱学。金属位置和衬底之间的相互作用将 用顺磁性金属离子的ESR波谱获得 衍生品。光谱研究将得到以下补充 取代潜在的氨基酸配体(组氨酸、半胱氨酸、 天冬氨酸和谷氨酸)。这个 突变蛋白将被用于序列特定的分配 将蛋白质配体连接到金属中心并作为作用的探针 这些金属离子在催化事件中发挥作用。身份和功能 该酶活性部位的其他氨基酸的含量将被测定 通过设计合成了炔基磷酸酯。这些 自杀底物与活性部位的亲核试剂发生共价反应 导致催化剂完全失活。重原子氧-18 利用野生型和突变体对快底物和慢底物的同位素效应 蛋白质将被用来确定过渡的分布 国家结构。X射线结晶学分析将在 努力确定褶皱的三维结构 蛋白。

The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the complex relationships between structure and function in biological systems. The primary focus of the present application is directed at the mechanism of catalysis and structure of the bacterial phosphotriesterase. This enzyme catalyzes the detoxification or organophosphate neurotoxins through the hydrolysis of phosphorus-oxygen and phosphorus-fluorine bonds. The phosphotriesterase catalyzes the hydrolysis of optimal substrates at the diffusion- controlled limit and a divalent cation is required for catalytic activity. The role of the essential metal ion will be probed by substitution of the native Zn2+ ion with a variety of spectroscopically active cations (Cd2+, Mn2+, Ni2+, Co2+, and Cu2+). The ligand environment of the metal sites will be addressed by 113Cd-NMR spectroscopy. Interactions between the metal sites and substrates will be obtained using ESR spectroscopy with the paramagnetic metal ion derivatives. The spectroscopic studies will be complemented by replacement of potential amino acid ligands (histidine, cysteine, aspartate, and glutamate) using site-directed mutagenesis protocols. The mutant proteins will be utilized for the sequence specific assignments of the protein ligands to the metal centers and as probes for the role these metal ions play in the catalytic events. The identity and function of other amino acids at the active site of this enzyme will be determined through the design and synthesis of alkynyl phosphate esters. These suicide substrates covalently react with active site nucleophiles and result in complete inactivation of catalysis. Heavy atom oxygen-18 isotope effects with fast and slow substrates using wild type and mutant proteins will be utilized to determine the distribution of transition state structures. An X-ray crystallographic analysis will begin in an effort to determine the three-dimensional structure of the folded protein.