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 a model 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 AMP-binding 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.
Skip Nav Destination
Article navigation
May 2005
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
April 26 2005
Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani
Rupak DATTA;
Rupak DATTA
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Ishita DAS;
Ishita DAS
1
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Banibrata SEN;
Banibrata SEN
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Anutosh CHAKRABORTY;
Anutosh CHAKRABORTY
2
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Subrata ADAK;
Subrata ADAK
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Chhabinath MANDAL;
Chhabinath MANDAL
†Division of Drug Design, Development and Molecular Modelling, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
Search for other works by this author on:
Alok K. DATTA
Alok K. DATTA
3
*Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
3To whom correspondence should be addressed (email alokdatta@iicb.res.in).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
October 13 2004
Revision Received:
December 15 2004
Accepted:
December 17 2004
Accepted Manuscript online:
December 17 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 387 (3): 591–600.
Article history
Received:
October 13 2004
Revision Received:
December 15 2004
Accepted:
December 17 2004
Accepted Manuscript online:
December 17 2004
Citation
Rupak DATTA, Ishita DAS, Banibrata SEN, Anutosh CHAKRABORTY, Subrata ADAK, Chhabinath MANDAL, Alok K. DATTA; Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani. Biochem J 1 May 2005; 387 (3): 591–600. doi: https://doi.org/10.1042/BJ20041733
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.