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PDBsum entry 1kic
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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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Enzyme-Substrate interactions in the purine-Specific nucleoside hydrolase from trypanosoma vivax.
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Authors
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W.Versées,
K.Decanniere,
E.Van holsbeke,
N.Devroede,
J.Steyaert.
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Ref.
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J Biol Chem, 2002,
277,
15938-15946.
[DOI no: ]
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PubMed id
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Abstract
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Nucleoside hydrolases are key enzymes in the purine salvage pathway of
Trypanosomatidae and are considered as targets for drug design. We previously
reported the first x-ray structure of an inosine-adenosine-guanosine preferring
nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (). Here we report the
2.0-A crystal structure of the slow D10A mutant in complex with the inhibitor
3-deaza-adenosine and the 1.6-A crystal structure of the same enzyme in complex
with a genuine substrate inosine. The enzyme-substrate complex shows the
substrate bound to the enzyme in a different conformation from 3-deaza-adenosine
and provides a snapshot along the reaction coordinate of the enzyme-catalyzed
reaction. The chemical groups on the substrate important for binding and
catalysis are mapped. The 2'-OH, 3'-OH, and 5'-OH contribute 4.6, 7.5, and 5.4
kcal/mol to k(cat)/K(m), respectively. Specific interactions with the exocyclic
groups on the purine ring are not required for catalysis. Site-directed
mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a
parallel aromatic stacking interaction involving Trp(83) and Trp(260). The pH
profiles of k(cat) and k(cat)/K(m) indicate the existence of one or more proton
donors, possibly involved in leaving group activation. However, mutagenesis of
the active site residues around the nucleoside base and an alanine scan of a
flexible loop near the active site fail to identify this general acid. The
parallel aromatic stacking seems to provide the most likely alternative
mechanism for leaving group activation.
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Figure 2.
Fig. 2. Structure of the D10A mutant of the IAG-NH from
T. vivax in complex with inosine. The inosine molecules located
in each active site of the IAG-NH dimer are shown as
ball-and-stick models, the calcium ions are depicted as blue
spheres. Amino acids 245-256 were excluded from the model.
Arrows indicate the position of the flexible loop containing
these amino acids in one of the subunits of the IAG-NH dimer.
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Figure 4.
Fig. 4. F[o]  F[c] map
around an inosine in one of the active sites of the D10A IAG-NH.
A, F[o] F[c] map
contoured at 3  . B, F[o]
F[c] map
contoured at 4.5 . The C-4'
endo envelope conformation of the ribose is shown.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2002,
277,
15938-15946)
copyright 2002.
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