Linked Life Data

11914097

Source:http://linkedlifedata.com/resource/pubmed/id/11914097

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
13
pubmed:dateCreated
2002-3-26
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
2
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4492-502
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:11914097-Alanine, pubmed-meshheading:11914097-Azides, pubmed-meshheading:11914097-Binding Sites, pubmed-meshheading:11914097-Carboxylic Acids, pubmed-meshheading:11914097-Catalysis, pubmed-meshheading:11914097-Chromatography, High Pressure Liquid, pubmed-meshheading:11914097-Chromatography, Thin Layer, pubmed-meshheading:11914097-Dose-Response Relationship, Drug, pubmed-meshheading:11914097-Humans, pubmed-meshheading:11914097-Hydrogen-Ion Concentration, pubmed-meshheading:11914097-Hydrolysis, pubmed-meshheading:11914097-Kinetics, pubmed-meshheading:11914097-Models, Chemical, pubmed-meshheading:11914097-Models, Molecular, pubmed-meshheading:11914097-Mutagenesis, Site-Directed, pubmed-meshheading:11914097-Mutation, pubmed-meshheading:11914097-Oligosaccharides, pubmed-meshheading:11914097-Pancreas, pubmed-meshheading:11914097-Pichia, pubmed-meshheading:11914097-Protein Binding, pubmed-meshheading:11914097-Recombinant Proteins, pubmed-meshheading:11914097-Salicylic Acids, pubmed-meshheading:11914097-Time Factors, pubmed-meshheading:11914097-alpha-Amylases
pubmed:year
2002
pubmed:articleTitle
Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't