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PDBsum entry 1kbb
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Mechanistic analyses of catalysis in human pancreatic alpha-Amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
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Authors
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E.H.Rydberg,
C.Li,
R.Maurus,
C.M.Overall,
G.D.Brayer,
S.G.Withers.
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Ref.
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Biochemistry, 2002,
41,
4492-4502.
[DOI no: ]
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PubMed id
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Abstract
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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.
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