Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

Abstract

The structure of the acylenzyme intermediate in the hydrolysis of the specific spin-label ester substrate methyl N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-tryptophan ate and its fluoro analogs catalyzed by alpha-chymotrypsin (EC 3.4.21.1) has been determined by electron nuclear double resonance (ENDOR) and molecular modeling methods. By a combination of kinetic and cryoenzymological methods, we have established conditions to stabilize the spin-labeled acylenzyme reaction intermediate. Proton ENDOR features specific for the substrate were assigned on the basis of specific deuteration. From the observed ENDOR shifts for protons and fluorines that correspond to principal hyperfine coupling components, the dipolar hyperfine coupling contributions were calculated to estimate electron-nucleus distances. With these dipolar separations as constraints, conformations of the substrate both free in solution and in the active site of alpha-chymotrypsin were determined by molecular graphics analysis. Comparison of the conformation of the bound substrate to that of the free substrate showed that formation of the acylenzyme requires significant torsional alteration in substrate structure. The structural relationships between active-site residues and the substrate in its ENDOR-assigned conformation are examined with respect to the requirements of stereoelectronic rules for formation and breakdown of the acylenzyme species.