Identification of Glutamic Acid 186 Affinity-Labeled by 2,3-Epoxypropyl α-D-Glucopyranoside in Soybean β-Amylase

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Abstract
Soybean β-amylase was modified with 2,3-epoxypropyl α-D-[U-14C]glucopyranoside ([14C]α-EPG), a radioactive affinity-labeling reagent for β-amylase, until it lost 95% of its enzyme activity. After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC. A radioactive peptide was finally isolated and its amino acid sequence was determined to be 181Leu-Gly-Pro-Ala-Gly-Glu186. Radioactivity derived from [14C]-α-EPG was found exclusively at Glu-186, the γ-carboxyl group of which is esterified with the affinity label. It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean β-amylase.