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Homology-Model-Guided Site-Specific Mutagenesis Reveals the Mechanisms of Substrate Binding and Product-Regulation of Adenosine Kinase from Leishmania donovani

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Title Homology-Model-Guided Site-Specific Mutagenesis Reveals the Mechanisms of Substrate Binding and Product-Regulation 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 Despite designating catalytic roles of Asp299 and Arg131 during the transfer of γ -phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty,Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591–600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study,
employing homology-model-guided site-specific protein mutagenesis, we showthat Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in Km (Ado) with a 1000-fold decrease in kcat/Km, implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance
of steric complementarity of the binding pocket. Use of 2�or 3�- eoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme–Ado complex, the binding energy (��GB) contribution of the formerwas greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding
studies revealed that, unlike the R131A mutant, which showed
abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered Km (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.
 
Date 2006
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1039/1/biojrupak2.pdf
Datta, Rupak and Das, Ishita and Sen, Banibrata and Chakraborty, Anutosh and Adak, Subrata and Mandal, Chhabinath and Dutta, Alok K (2006) Homology-Model-Guided Site-Specific Mutagenesis Reveals the Mechanisms of Substrate Binding and Product-Regulation of Adenosine Kinase from Leishmania donovani. Biochemical Journal, 394. pp. 35-42.
 
Relation http://dx.doi.org/10.1042/BJ20051513
http://www.eprints.iicb.res.in/1039/