title: The mechanism of salivary amylase hydrolysis: role of residues at subsite S2'.
abstract: Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in maltose as the leaving group. The active site of these amylases harbors three aromatic residues Trp59, Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties. We hypothesized that Tyr151, located at the S2' subsite, may influence the size of the leaving group. Therefore, using a baculovirus expression system, we generated a mutant Y151M in which the tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific activity, K(m), rate of hydrolysis, and the product distribution for Y151M were distinctly different from those of the wild-type enzyme using starch and oligosaccharides as substrates. The mutant enzyme Y151M consistently produced glucose as the minimal leaving group and exhibited a twofold increase in K(m). These results suggest that the stacking interaction at subsite S2' in the wild type plays a role in hydrolysis.
authors.affiliation: Dental Research Center, University of Medicine and Dentistry, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
authors.forename: Prasunkumar J
authors.initials: PJ
authors.lastname: Mishra
authors.forename: Chandran
authors.initials: C
authors.lastname: Ragunath
authors.forename: Narayanan
authors.initials: N
authors.lastname: Ramasubbu
mesh_terms: D000681:Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D005947:Glucose / Q000096:biosynthesis; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000096:biosynthesis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D012469:Salivary Glands / Q000201:enzymology*; D013213:Starch / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D008320:Maltose; D013213:Starch; D000681:Amylases; D005947:Glucose