Abstract
The substrate binding ability of Taka-amylase A (TAA) was evaluated by chemical modification, affinity gel electrophoresis and affinity chromatography methods.. Compared with modifications of the amino group with o-phthalaldehyde (OPA) and 2, 4, 6-trinitrobenzenesulfonic acid (TNBS), modification of the Trp residue with N-bromosucciniimide (NBS) caused a strong and rapid inactivation of TAA. NBS-modified TAA greatly impaired the affinity to substrate soluble starch. Based on these results and previous results of isolation of substrate-binding peptides, all of which contained Trp residues in the sequence [Y. SASAKI et al.: Biosci. Biotech. Biochem., 61, 1840-1843 (1997)], it was strongly suggested that Trp residues in the TAA molecule played an important role in substrate binding and subsequently in the catalytic activity. Moreover, affinity analyses, which were useful for the evaluation of enzyme affinity to substrates or its analogs having low susceptibility to TAA in the activity measurement, indicated that TAA could recognize the dextran molecule as the substrate analog.
