Mechanistic Studies on Dinuclear Hydrolytic Catalysts

在这个由无机、生物无机和有机金属化学项目支持的项目中，犹他州立大学的理查德·c·霍尔兹博士将研究多核水解酶中单个金属离子在催化反应中所起的作用。这项工作的初始阶段将围绕着来自水解弧菌的双核锌（II）氨基肽酶的研究展开，该酶的质粒可用于在大肠杆菌中过表达该酶。该方法将涉及用光谱活性Co(II), Ni（II）或Cu（II）离子取代酶中的单个锌离子，这些离子已经被证明也显示出增强的酶活性。在一些研究中，镉（II）也将被用作替代品。方法包括紫外-可见，核磁共振，EPR和EXAFS光谱，以及稳态动力学。需要考虑的具体问题包括：1)与其他金属离子取代后获得超活性的配体的性质和几何形状是什么？2)每种金属中心在催化中起什么具体作用？3)底物如何与双核活性位点相互作用？4)一个或两个金属离子是否作为路易斯酸结合并激活水分子进行水解反应？含有一种以上金属离子的水解酶（多核酶）直到最近才被证实存在。这些酶发挥催化作用的机制目前尚不清楚。本研究旨在确定单个金属离子在催化反应中所起的作用。这一结果可能导致可用于生物技术的合成酶的开发。

In this project, which is supported by the Inorganic, Bioinorganic and Organometallic Chemistry Program, Dr. Richard C. Holz of Utah State University will undertake a study of the roles that the individual metal ions in multinuclear hydrolytic enzymes play in their catalytic reactions. The initial phase of the work will be built around studies of the dinuclear Zn(II) aminopeptidase from Vibrio proteolytica, for which a plasmid is available that can be used to overexpress the enzyme in E. coli. The approach will involve substitution of individual zinc ions in the enzyme with spectroscopically active Co(II), Ni(II) or Cu(II) ions, which have already been demonstrated to also show enhanced enzymatic activity. Cadmium(II) will also be used for substitution in some studies. Methods to be used include UV-VIS, NMR, EPR and EXAFS spectroscopies, as well as steady state kinetics. Specific questions to be considered include: 1) What is the nature and geometry of the ligands upon substitution with other metal ions such that hyperactivity is obtained. 2) What specific role does each metal center play in catalysis? 3) How do substrates interact with the dinuclear active site? and 4) Does one or both metal ions serve as a Lewis acid to bind and activate a water molecule for the hydrolysis reaction? Hydrolytic enzymes that contain more than one metal ion (multinuclear enzymes) have been demonstrated to exist only recently. The mechanisms by which these enzymes effect catalysis are presently unknown. This research is designed to determine the role that the individual metal ions play in the catalytic reaction. The results may lead to the development of synthetic enzymes that can be used in biotechnology.