Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

Datta, Rupak ; Das, Ishita ; Sen, Banibrata ; Chakraborty, Anutosh ; Adak, Subrata ; Mandal, Chhabinath ; Datta, 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. ISSN 0006-2936

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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.

Item Type:Article
Source:Copyright of this article belongs to Biochemical Society.
Keywords:Active Site; Adenosine Kinase; Leishmania donovani; Purine Salvage; Ribokinase; Site-directed Mutagenesis
ID Code:86518
Deposited On:10 Mar 2012 12:56
Last Modified:10 Mar 2012 12:56

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