This proposal outlines experiments that are designed to provide considerable insight into: (B) the active-site structure and dynamics. (B) the mechanisms and forces involved in the binding of inhibitors, and (C) the catalytic mechanism of serine proteases. Our approach emphasizes 15 N NMR spectroscopy to gain specific information about the properties and the environment of the active- site histidine. We will employ, however, other one-dimensional and two-dimensional NMR techniques whenever desirable and appropriate.
Specific aims of this proposal are: 1. To examine and characterize the properties and the environment of His-57 in complexes formed between alpha-lytic protease and other serine proteases, and the following classes of inhibitors: (a) peptide-boronic acids, (b) peptide-aldehydes, (c) peptide fluoromethylketones, (d) isotoic anhydrides, (e) protein-protease inhibitors. 2. To determine the molecular basis behind the phenomenon of slow-binding inhibition exhibited by the peptide-boronic acids. 3. To use 15N NMR spectroscopy to examine His-57 of alpha-lytic protease in the crystalline state to gain a direct comparison of the properties and environment of His-57 between the crystal and solution structures. 4. To probe the structure of enzyme-substrate complexes. 5. To extend our 15N NMR approach for studying histidyl residues to other serine proteases.
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