The structural and dynamical basis of enzyme action, primarily of proteolytic enzymes, will be investigated by (i) cryokinetic methods to stabilize true enzyme reaction intermediates; (ii) electron nuclear double resonance spectroscopy to determine the structure and conformation of substrates free in solution and bound in enzymic active sites of catalytically competent reaction intermediates; (iii) molecular graphics and modeling to assign stereochemical relationships of enzyme-substrate interactions based on spectroscopically determined data; (iv) interpreta- tion of structural data according to stereoelectronic principles; and (v) assessment of the relationships of dynamical motion of protein residues to enzyme catalytic function by computer based molecular dynamics simulations. The methods are designed to determine local structure of enzyme active site residues and of substrates in kinetically competent reaction intermediates, to assess the influence of protein dynamical motion in torsionally altering substrate structure to allow tight, specific binding in the active site, and to assess the structural and electronic basis of enzyme action on a detailed molecular level. The methods to be applied will yield data to assign three-dimensional structure and conformation of catalytically competent reaction intermediates of enzymes in a manner that cannot be achieved at present with other physical methods.