Questions pertaining to the structure, dynamics, and catalytic mechanism of serine proteases will be addressed using nitrogen-15 and carbon 13 NMR. In general, specifically labelled enzyme derivatives, obtained through biosynthetic enrichment techniques, will be employed in order to facilitate detection and resolution of desired signals, and the enriched enzymes will be studied in both solution and solid states. The solid state studies will include recording both powder spectra, and high resolution spectra through magic angle sample spinning (MASS). Both lyophilized powder and crystalline forms of the enzyme will be examined.
Specific aims of this proposal include: (1) Characterizing the properties of the active site functional groups, with special attention given to evaluating the significance of these properties for catalysis. These properties include microscopic pka's, hydrogen-bond interactions, tautomeric structure, functional group dynamics, and dynamics of proton exchange. (2) Determination of the correspondance between the crystallographically determined structure and that of the equilibrium structure in solution. (3) Determination of the effect of inhibitor binding on the properties of the active site functional groups. (4) Development of methods for using NMR to directly study enzyme-substrate complexes.
| Bachovchin, W W (1986) 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry 25:7751-9 |
| Bachovchin, W W (1985) Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme. Proc Natl Acad Sci U S A 82:7948-51 |
