Questions pertaining to the structure, dynamics, and catalytic mechanism of serine proteases will be addressed using nitrogen-15 and carbon-13 nuclear magnetic resonance spectroscopy. In general, specifically labeled enzymes, obtained through biosynthetic isotopic enrichment techniques, will be employed to facilitate detection and resolution of the desired signals. The labeled enzymes will be studied in both the solution and crystalline states. High resolution MNR spectra of enzyme crystals will be obtained using magic angle sample spinning (MASS).
Specific aims of this proposal are: (1) To characterize the hydrogen bonding interactions across the Asp-His-Ser Triad in Alpha-lytic protease, both for the resting enzyme and for a series of inhibitor complexes. This includes confirming our preliminary results which indicate that histidine and serine are strongly hydrogen bonded in the resting enzyme in solutions, and that the Asp-His hydrogen bond becomes disrupted in response to specific events. (2) To gain a direct comparison of the Asp-His-Ser interactions between crystals and solutions, both for the resting enzyme and for several inhibitor complexes. (3) To characterize the proton exchange reactions involving the catalytic triad. This includes dissecting and separately evaluating exchange between the triad and solvent water from proton exchange reactions occurring among the functional groups comprising the triad. (4) To characterize the Asp-His-Ser interactions in subtilisin and compare them to those of Alpha-lytic protease. (5) To directly observe and study the functional groups of serine and aspartic acid in C13 NMR spectra. (6) To explore the feasibility of obtaining N15 NMR information about histidyl residue in proteins at natural abundance levels of nitrogen-15.
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