Affinity Labeling of Soybean β-Amylase with 2′,3′-Epoxypropyl α-D-Glucopyranoside

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Abstract
The synthesized 2′,3′-epoxypropyl α-D-glucopyranoside (α-EPG) inactivated soybean β -amylase completely. The incorporation of α-EPG into the enzyme at 92% inactivation was 1.1 mol per mol of enzyme, as determined by using 14 C-labeled α-EPG. The inactivation obeyed saturation kinetics of a two-step mechanism. The dissociation constant of α-EPG-enzyme complex and the rate constant of the irreversible inactivation step were estimated to be 119 mM and 1.14×10 −3 s −1 , respectively. α-Cyclodextrin, a competitive inhibitor of this enzyme, protected the enzyme against the inactivation by α-EPG in a competitive manner. This suggests that α-EPG binds to the active site of the enzyme. The above results indicate that α-EPG acts on soybean β -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 α-EPG.