APPLICATION OF SECONDARY ALPHA-DEUTERIUM KINETIC ISOTOPE EFFECTS TO STUDIES OF ENZYME CATALYSIS . GLYCOSIDE HYDROLYSIS BY LYSOZYME AND BETA-GLUCOSIDASE

Secondary kinetic isotope effects (α-deuterium) have been explored as a method for delineating the nature of the transition state in enzyme-catalyzed reactions. Hen egg-white lysozyme and almond β-glucosidase were chosen as enzymes for these experiments. Initially, model studies were performed on the cleavage of phenyl glucosidic bonds in acid (SnI mechanism) and in base (Sn2 mechanism). The values obtained (kH/kD = 1.13 for Sn1 mechanism, kH/kD = 1.03 for Sn2 mechanism) were in the anticipated range. The value obtained for β-glucosidase-catalyzed hydrolysis of phenyl β-D-glucopyranoside was kH/kD = 1.01, and this suggests a displacement mechanism for this enzyme. Such a result is in agreement with recent chemical evidence. For application of the method to studies of the lysozyme mechanism, the disaccharide phenyl 4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-β-D-glucopyranoside was synthesized by enzymatic transglycosylation using 3H- and 14C-labeled phenyl glucosides (C1-H and C1-d, respectively). The value of kH/kD obtained for the lysozyme-catalyzed hydrolysis of this aryl disaccharide glycoside was determined to be 1.11. This result indicates considerable carbonium ion character in the transition state of the enzyme-catalyzed reaction. It was found that this result held over the pH range 3.1-8.3 as well as close to the pH optimum. © 1969, American Chemical Society. All rights reserved.