ESR probing of macromolecules: Spin label study of the conformational integrity and transitions in modified α-chymotrypsin

The spin-labeling method has been used to probe the effect of specific modification of α-chymotrypsin on the conformational integrity of this enzyme's active center. Labels on either the serine 195 hydroxyl or methionine 192 thioether or both provide spectral data which indicate the following: (1) alkylation of one or both of the methionines in α-chymotrypsin with all reagents tried causes a loss of "structure" of the active center; (2) conversely, oxidation of these residues has little effect; and (3) this loss of structure has little effect on the poising of the catalytically functional groups, but diminishes the productivity of substrate binding via steric inhibition. Associated with this latter point is evidence that methionine 192 moves in response to the ionization of aspartic acid 194 and isoleucine 16. The relationship of this evidence to the crystallographic model of the enzyme and its catalytic operation is discussed.