Affinity labeling of soybean beta-amylase with 2',3'-epoxypropyl alpha-D-glucopyranoside.

The synthesized 2',3'-epoxypropyl alpha-D-glucopyranoside (alpha-EPG) inactivated soybean beta-amylase completely. The incorporation of alpha-EPG into the enzyme at 92% inactivation was 1.1 mol per mol of enzyme, as determined by using 14C-labeled alpha-EPG. The inactivation obeyed saturation kinetics of a two-step mechanism. The dissociation constant of alpha-EPG-enzyme complex and the rate constant of the irreversible inactivation step were estimated to be 119 mM and 1.14 X 10(-3)s-1, respectively. alpha-Cyclodextrin, a competitive inhibitor of this enzyme, protected the enzyme against the inactivation by alpha-EPG in a competitive manner. This suggests that alpha-EPG binds to the active site of the enzyme. The above results indicate that alpha-EPG acts on soybean beta-amylase as an affinity labeling reagent. It was also shown that an essential SH group near the active site, but not the catalytic one, scarcely participated in the inactivation by alpha-EPG.