The determination of the stereochemistry and electronic structure of substrates and active site residues in reaction intermediates of enzymes stabilized by cryosolvent methods is proposed. Reaction intermediates stabilized at subzero temperatures in organic-aqueous cosolvent mixtures will be prepared for spectroscopic studies by EPR and ENDOR methods. The radial separation between paramagnetic sites and protons and other nuclides in the vicinity of the paramagnetic sites will be determined on the basis of the EPR and ENDOR absorptions of substrates and catalytically essential active site residues. These results will then be employed to assign the exact catalytically productive configurations of substrates bound in the active site of enzymes through application of computer controlled molecular graphics techniques. The methods are designed to determine local structure of enzymic active sites and of substrates in kinetically competent reaction intermediates to assess the structural and electronic basis of enzyme function on a detailed molecular level.
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