Examination of Aglycone-Binding Site of Human Salivary α-Amylase by Means of Transglycosylation Reactions

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Abstract
The active site of human salivary α-amylase is composed of tandem subsites (S3, S2, S1, S1′, S2′, etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1′. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1′, S2′), respectively. The features of the aglycone-binding site of human salivary α-amylase were examined by means of transglycosylation reaction using phenyl α-maltoside (GGΦ: G-G-Φ) and its derivatives (GAGΦ: G-AG-Φ, GCGΦ: G-CG-Φ, AGGΦ: AG-G-Φ, and CGGΦ: CG-G-Φ) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl α-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAGΦ occurred more effectively than to GGΦ. Transfers to GCGΦ and CGGΦ were less than to GGΦ and very little transfer to AGGΦ occurred. When transglycosylation reaction occurs, the acceptors should approach the aglycone-binding site (S1′, S2′) and their reducing-end and non-reducing-end glucose residues interact with S2′ and S1′, respectively. The results obtained suggested that AG and CG residues were hardly bound to S1′ owing to the protonated amino group and carboxylate groups and that a negative charge of an acidic amino acid residue of the enzyme was near to the C-6 hydroxy group of the glucose residue bound to S2′

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