title: Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase.
abstract: Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.
authors.affiliation: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia , Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
authors.forename: Sami
authors.initials: S
authors.lastname: Caner
authors.affiliation: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Emily
authors.initials: E
authors.lastname: Kwan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia , Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
authors.forename: Chunmin
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia , Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Brayer
authors.affiliation: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.|Department of Biochemistry and Molecular Biology, University of British Columbia , Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D013213:Starch; D000516:alpha-Amylases