title: Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
abstract: The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Edwin H
authors.initials: EH
authors.lastname: Rydberg
authors.forename: Chunmin
authors.initials: C
authors.lastname: Li
authors.forename: Robert
authors.initials: R
authors.lastname: Maurus
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.forename: Gary D
authors.initials: GD
authors.lastname: Brayer
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000409:Alanine / Q000737:chemistry; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology*; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012459:Salicylates / Q000494:pharmacology; D013997:Time Factors; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001386:Azides; D002264:Carboxylic Acids; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012459:Salicylates; C019193:maltopentaose; D000516:alpha-Amylases; D000409:Alanine