Identification of glutamic acid 186 affinity-labeled by 2,3-epoxypropyl alpha-D-glucopyranoside in soybean beta-amylase.
Soybean beta-amylase was modified with 2,3-epoxypropyl alpha-D-[U-14C]glucopyranoside ([14C]alpha-EPG), a radioactive affinity-labeling reagent for beta-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]-alpha-EPG was found exclusively at Glu-186, the gamma-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 beta-amylase.
Nitta
Y
Y
Laboratory of Biophysical Chemistry, College of Agriculture, University of Osaka Prefecture.
Isoda
Y
Y
Toda
H
H
Sakiyama
F
F
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000681:Amylases / Q000032:analysis*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005960:Glucosides / Q000138:chemical synthesis; D005971:Glutamates / Q000032:analysis*; D006027:Glycosides; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases; D011489:Protein Denaturation; D013025:Glycine max / Q000201:enzymology*; D001614:beta-Amylase / Q000032:analysis*
D000345:Affinity Labels; D005960:Glucosides; D005971:Glutamates; D006027:Glycosides; C007607:2',3'-epoxypropylglucopyranoside; D000681:Amylases; D001614:beta-Amylase; D010447:Peptide Hydrolases