title: Roles of the aromatic residues conserved in the active center of Saccharomycopsis alpha-amylase for transglycosylation and hydrolysis activity.
abstract: The molecular structure of Saccharomycopsis fibuligera alpha-amylase was predicted by a homology-based modeling technique, and the amino acid residues composing the active site were displayed with color codes according to their order of conservation. We noticed two highly conserved aromatic residues located in the active center, tyrosine 83 (Y83) and tryptophan 84 (W84), and examined their roles in catalytic activity by site-directed mutagenesis. The W, leucine (L), and asparagine (N) mutants at Y83 and the L mutant at W84 showed remarkable enhancement of transglycosylation activity and complementary decreases in native hydrolysis activity. The phenylalanine (F) mutant at Y83 and the F and Y mutants at W84 only decreased hydrolysis activity. Mechanistic and kinetic studies of these mutants using a reducing-end-blocked substrate and a hydrolysis-specific substrate revealed a probable transglycosylation mechanism and critical contributions of the 83rd and 84th aromatic residues to efficient hydrolysis. Given that aromatic residues stack against the faces of sugars, we assumed that Y83 and, presumably, W84 play roles in the binding of oligosaccharide substrates through the stacking interaction and in the indirect fixation of the catalytic water molecule through hydrogen bonding with the hydroxyl of the bound substrates. Mutations to nonaromatic residues could cause slight changes in the binding topology of substrates to favor transglycosylation over hydrolysis.
authors.affiliation: National Institute of Bioscience and Human-Technology, Ibaraki, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Matsui
authors.forename: S
authors.initials: S
authors.lastname: Yoneda
authors.forename: K
authors.initials: K
authors.lastname: Ishikawa
authors.forename: S
authors.initials: S
authors.lastname: Miyairi
authors.forename: S
authors.initials: S
authors.lastname: Fukui
authors.forename: H
authors.initials: H
authors.lastname: Umeyama
authors.forename: K
authors.initials: K
authors.lastname: Honda
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D006031:Glycosylation; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012442:Saccharomycopsis / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D014364:Tryptophan; D000516:alpha-Amylases