Chemical Modification of Sulfhydryl Groups in Soybean β-Amylase

Abstract

The behavior of SH groups of soybean β-amylase was investigated by chemical modification. Two SH groups out of a total of five in the native enzyme reacted with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB), iodoacetamide or monoiodoacetate at high ionic strength, accompanied by inactivation of the enzyme. At low ionic strength, only one SH group was accessible to DTNB without inactivation. By using the reaction with iodoacetamide, the dissociation constants of these two SH groups and rate constants for the reaction were determined. The most reactive SH group, which was independent of the inactivation, reacted with monoiodoacetate 185 times faster than the essential SH group. Next, selective modification of one SH group with monoiodoacetate was carried out. The modified enzyme, having activity equal to that of the native enzyme, was purified by ion exchange column chromatography. It was successively treated with DTNB or iodoacetamide in order to achieve selective modification of the essential SH group. The enzyme modified further with DTNB was fully reactivated by 2-mercaptoethanol treatment. It also recovered 65% of the original enzymatic activity on treatment with cyanide but only 7% with sulfite.
Maltose and cyclohexadextrin protected the essential SH group from modification, the former being more effective. The ultraviolet absorption spectrum of soybean β-amylase was changed by modification of the essential SH group, and the spectrum was also changed by the binding of maltose. The change induced by maltose was influenced by modification of the essential SH group. It was concluded that the essential SH group of soybean β-amylase does not participate in the catalysis, but is situated near the binding site of maltose.