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Title
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X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases.
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Authors
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B.Strokopytov,
D.Penninga,
H.J.Rozeboom,
K.H.Kalk,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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Biochemistry, 1995,
34,
2234-2240.
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PubMed id
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Abstract
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Crystals of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans
strain 251 were soaked in buffer solutions containing the pseudotetrasaccharide
acarbose, a strong amylase- and CGTase inhibitor. The X-ray structure of the
complex was elucidated at 2.5-A resolution with a final crystallographic R value
of 15.8% for all data between 8.0 and 2.5 A. Acarbose is bound near the
catalytic residues Asp229, Glu257, and Asp328. The carboxylic group of Glu257 is
at hydrogen bonding distance from the glycosidic oxygen in the scissile bond
between the B and C sugars (residue A is at the nonreducing end of the
inhibitor). Asp328 makes hydrogen bonds with the 4-amino-4,6-dideoxyglucose
(residue B), and Asp229 is in a close van der Waals contact with the C1 atom of
this sugar. From this we conclude that in CGTase Glu257 acts as the proton donor
and Asp229 serves as the general base or nucleophile, while Asp328 is involved
in substrate binding and may be important for elevating the pKa of Glu257. On
the basis of these results it appears that the absence of the C6-hydroxyl group
in the B sugar is responsible for the inhibitory properties of acarbose on
CGTase. This suggests that the C6-hydroxyl group of this sugar plays an
essential role in the catalytic mechanism of CGTase.(ABSTRACT TRUNCATED AT 250
WORDS)
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