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.
