ESR PROBING OF MACROMOLECULES - FUNCTION AND OPERATION OF STRUCTURAL UNITS WITHIN ACTIVE SITE OF ALPHA-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. © 1969.