ESR probing of macromolecules: Function and operation of structural units within the active site of α-chymotrypsin

The spin-labelled p-nitrophenyl esters have been prepared from N-(2,2,6,6-tetra-methyl-1-oxyl-4-piperidinyl) succinamic acid, O-(2,2,6,6-tetramethyl-1-oxyl-4-piperidinyl) hydrogen succinate, and maleate. These esters are used to acylate α-chymotrypsin (α-ChT). The correlation of the changes in the electron spin resonance (ESR) spectra with (a) the structure of the substrate, (b) the presence or absence of indole, and (c) structural modifications of the enzyme (oxidation of Met₁₈₀ and/or Met₁₉₂) identifies the aryl binding region (Met₁₉₂) and amide binding region (Met₁₈₀). The correlation of the above changes with the rate of deacylation (k₃) indicates a cooperative action-reaction link between these two regions and the role this link plays in the overall hydrolytic process. A stepwise, operational sequence is postulated.