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Title
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The active center of a mammalian alpha-amylase. Structure of the complex of a pancreatic alpha-amylase with a carbohydrate inhibitor refined to 2.2-A resolution.
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Authors
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M.Qian,
R.Haser,
G.Buisson,
E.Duée,
F.Payan.
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Ref.
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Biochemistry, 1994,
33,
6284-6294.
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PubMed id
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Abstract
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An X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (EC
3.2.1.1) that was soaked with acarbose (a pseudotetrasaccharide alpha-amylase
inhibitor) showed electron density corresponding to five fully occupied subsites
in the active site. The crystal structure was refined to an R-factor of 15.3%,
with a root mean square deviation in bond distances of 0.015 A. The model
includes all 496 residues of the enzyme, one calcium ion, one chloride ion, 393
water molecules, and five bound sugar rings. The pseudodisaccharide acarviosine
that is the essential structural unit responsible for the activity of all
inhibitors of the acarbose type was located at the catalytic center. The
carboxylic oxygens of the catalytically competent residues Glu233 and Asp300
form hydrogen bonds with the "glycosidic" NH group of the acarviosine group. The
third residue of the catalytic triad Asp197 is located on the opposite side of
the inhibitor binding cleft with one of its carbonyl oxygens at a 3.3-A distance
from the anomeric carbon C-1 of the inhibitor center. Binding of inhibitor
induces structural changes at the active site of the enzyme. A loop region
between residues 304 and 309 moves in toward the bound saccharide, the resulting
maximal mainchain movement being 5 A for His305. The side chain of residue
Asp300 rotates upon inhibitor binding and makes strong van der Waals contacts
with the imidazole ring of His299. Four histidine residues (His101, His201,
His299, and His305) are found to be hydrogen-bonded with the inhibitor. Many
protein-inhibitor hydrogen bond interactions are observed in the complex
structure, as is clear hydrophobic stacking of aromatic residues with the
inhibitor surface. The chloride activator ion and structural calcium ion are
hydrogen-bonded via their ligands and water molecules to the catalytic residues.
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