Studies on the Active Site of Taka-amylase A: Its Action on Phenyl Maltooligosides with a Charge at Their Non-Reducing-Ends1

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Abstract
Five modified maltooligosaccharides, phenyl 0-6-amino-6-deoxy-α-D-glucopyranosyl-(1↠4)-0-α-D-glucopyranosyl- (1↠4)-0-α-D- glucopyranosyl -(1↠4)-α-D- glucopyranoside (AG4P), phenyl O-(α-D-glucopyranosyluronic acid)-(l↠4)-0-α-D-gluco-pyranosyl-(l ↠4)-0-α-D-glucopyranosyl-(l↠4)-α-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-α-D-glucopyranosyl-(l ↠4)-0-α-D-glucopyranosyl-(l ↠4)-0-α-D- glucopyranosyl-(l ↠4)-0-α-D-glucopyranosyl-(l↠4)-α-D-glucopyranoside (AG5P), phenyl O-(α-D-glucopyranosyluronic acid)-(l↠4)-O-α-D-glucopyranosyl-(l↠4)-0-α-D-glucopyranosyl-(l↠4)-0-α-D-glucopyranosyl-(l↠4)-α-D-glucopyranoside (CG5P), and phenyl 0-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(l↠4)-O-α-D-gluco-pyranosyl-(l↠4)O-α-D-glucopyranosyl-(l↠4)-α-D-glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Asper-gillus oryzae]. Phenyl α-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl α-maltoside (G2P). While G2P, phenyl α-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7 : 63 : 30. G4P, phenyl α-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1 :20 :73 : 6, but AG5P was almost completely hydrolyzed to modified malto-triose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively. When FG4P and G4P were hydrolyzed in the pH range of 4.5–6.0, the molar ratio of the hydrolysis products of G4P remained almost constant. However, that in the case of FG4P changed with pH, i.e., GP was predominantly formed at lower pHs, while the formation of G2P increased at higher pHs. These results suggest the presence of acidic amino acids at subsites S3 and S4 in the active site of TAA that interact with amino or pyridylamino groups of the substrates