title: Structural advantage of sugar beet α-glucosidase to stabilize the Michaelis complex with long-chain substrate.
abstract: The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.
authors.affiliation: From the Research Faculty of Agriculture.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Tagami
authors.affiliation: Graduate School of Life Science, and.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Yamashita
authors.affiliation: From the Research Faculty of Agriculture, okuyama@abs.agr.hokudai.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.affiliation: From the Research Faculty of Agriculture.
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.affiliation: Graduate School of Life Science, and Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.affiliation: From the Research Faculty of Agriculture, kimura@abs.agr.hokudai.ac.jp.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D001483:Base Sequence; D027461:Beta vulgaris / Q000201:enzymology*; D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain; D003460:Crystallization; D005947:Glucose / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D009844:Oligosaccharides; C016549:maltohexaose; D000520:alpha-Glucosidases; D005947:Glucose
keywords: Carbohydrate Structure; Crystal Structure; Enzyme Kinetics; Glycoside Hydrolase; Transition State Analog