Studies on the substrate specificity of Taka-amylase A. XII. Investigation of the active site of Taka-amylase A by examining the properties of p-phenylazobenzoyl Taka-amylase A.
1. When p-phenylazobenzoyl Taka-amylase A (PhAB-TAA) was incubated at pH 6.5 with hydroxylamine for 3 hr at 20degrees, some of the p-phenylazobenzoyl residues that had been introduced into Taka-amylase A (TAA) [1, 4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1, Aspergillus oryzae] were liberated as a hydroxamic acid, and the activity pattern of PhAB-TAA changed to that of intact TAA. This result suggested that the p-phenylazobenzoyl residues liberated had been bound to the tyrosyl residue located near the active site in the enzyme. 2. The transferase action of TAA or PhAB-TAA was studied using phenyl alpha-maltoside as a substrate and maltotritol as an acceptor. Unlike intact TAA, PhAB-TAA was not able to transfer the maltose residue to maltotritol, and this suggested that the p-phenylazobenzoyl residue was located near one of the aglycone-binding subsites, causing steric hindrance.
D000681:Amylases / Q000378:metabolism; D001565:Benzoates; D001665:Binding Sites; D002849:Chromatography, Gas; D007700:Kinetics; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014166:Transferases / Q000378:metabolism
D001565:Benzoates; D014166:Transferases; D000681:Amylases