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

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.

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    Abstract

    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.

    Item Type: Article
    URI: http://www.eprints.iicb.res.in/id/eprint/1039
    Subjects: Structural Biology & Bioinformatics
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Mr Shyamal Nath
    Date Deposited: 27 Dec 2011 16:56
    Last Modified: 03 Feb 2012 11:43
    Official URL: http://dx.doi.org/10.1042/BJ20051513
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