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Mutational Analysis of the Active-Site Residues Crucial for Catalytic Activity of Adenosine Kinase from Leishmania donovani

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Title Mutational Analysis of the Active-Site Residues Crucial for Catalytic Activity of Adenosine Kinase from Leishmania donovani
 
Creator Datta, Rupak
Das, Ishita
Sen, Banibrata
Chakraborty, Anutosh
Adak, Subrata
Mandal, Chhabinath
Dutta, Alok K
 
Subject Structural Biology & Bioinformatics
 
Description Leishmania donovani adenosine kinase (LdAdK) plays a pivotal
role in scavenging of purines from the host. Exploiting interspecies
homology and structural co-ordinates of the enzyme from
other sources,we generated amodel of LdAdK that led us to target
several amino acid residues (namely Gly-62, Arg-69, Arg-131 and
Asp-299). Replacement of Gly-62 with aspartate caused a drastic
reduction in catalytic activity, with decreased affinity for either
substrate. Asp-299 was found to be catalytically indispensable.
Mutation of either Arg-131 or Arg-69 caused a significant reduction
in kcat. R69A (Arg-69→Ala) and R131A mutants exhibited
unaltered Km for either substrate, whereas ATP Km for R69K
increased 6-fold. Importance of both of the arginine residues was
reaffirmed by the R69K/R131A double mutant, which exhibited
approx. 0.5% residual activity with a large increase in ATP
Km. Phenylglyoxal, which inhibits the wild-type enzyme, also
inactivated the arginine mutants to different extents. Adenosine
protected both of the Arg-69 mutants, but not the R131A variant,
from inactivation. Binding experiments revealed that the AMPbinding
property of R69K or R69A and D299A mutants remained
largely unaltered, but R131A and R69K/R131A mutants lost
their AMP binding ability significantly. The G62D mutant did
not bind AMP at all. Free energy calculations indicated that
Arg-69 and Arg-131 are functionally independent. Thus, apart
from the mandatory requirement of flexibility around the diglycyl
(Gly-61–Gly-62) motif, our results identified Asp-299 and Arg-
131 as key catalytic residues, with the former functioning as the
proton abstractor from the 5�-OH of adenosine, while the latter
acts as a bidentate electrophile to stabilize the negative charge
on the leaving group during the phosphate transfer.Moreover, the
positive charge distribution of Arg-69 probably helps in maintaining
the flexibility of the α-3 helix needed for proper domain
movement. These findings provide the first comprehensive biochemical
evidence implicating the mechanistic roles of the functionally
important residues of this chemotherapeutically exploitable
enzyme.
Key words: active site, adenosine kinase, Leishmania donovani,
purine salvage, ribokinase, site-directed mutagenesis.
 
Date 2005
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1036/1/biojrupak.pdf
Datta, Rupak and Das, Ishita and Sen, Banibrata and Chakraborty, Anutosh and Adak, Subrata and Mandal, Chhabinath and Dutta, Alok K (2005) Mutational Analysis of the Active-Site Residues Crucial for Catalytic Activity of Adenosine Kinase from Leishmania donovani. Biochemical Journal, 387. pp. 591-600.
 
Relation http://dx.doi.org/
http://www.eprints.iicb.res.in/1036/