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PDBsum entry 1kgu
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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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Probing the role of the chloride ion in the mechanism of human pancreatic alpha-Amylase.
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Authors
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S.Numao,
R.Maurus,
G.Sidhu,
Y.Wang,
C.M.Overall,
G.D.Brayer,
S.G.Withers.
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Ref.
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Biochemistry, 2002,
41,
215-225.
[DOI no: ]
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PubMed id
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Abstract
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Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family
involved in the degradation of starch. Some members of this family, including
HPA, require chloride for maximal activity. To determine the mechanism of
chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q)
were made in which residues in the chloride ion binding site were replaced.
Mutations in this binding site were found to severely affect the ability of HPA
to bind chloride ions with no binding detected for the R195 and R337 mutant
enzymes. X-ray crystallographic analysis revealed that these mutations did not
result in significant structural changes. However, the introduction of these
mutations did alter the kinetic properties of the enzyme. Mutations to residue
R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward
starch and shifted the pH optimum to a more basic pH. Interestingly, replacement
of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer
required chloride for catalysis and has a pH profile similar to that of
wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme
that had much lower binding affinity for chloride but still required chloride
for maximal activity. We propose that the chloride is required to increase the
pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the
presence of R337, a positively charged residue.
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