Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani
Metadata of CSIR Papers
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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
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| Creator |
Datta, R
Das, I Sen, B Chakraborty, A Adak, S Mandal, C Datta, AK |
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| Subject |
Biochemistry & Molecular Biology
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| Description |
Despite designating catalytic roles of Asp(299) and Arg(131) during the transfer of gamma-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak. Mandal and A. K. Datta (2005) Bio-chem. 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 muta-genesis, we show that Asp(16) is indispensable, since its replacement with either valine or arginine resulted in a > 200-fold increase in K-m (Ado) witha 1000-fold decrease in k(cat)/K-m implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp(16) in the maintenance of steric complementarity of the binding pocket. Use of Tor 3'-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme-Ado complex, the binding energy (Delta G(B)) contribution of the former was greater than the latter, Suggesting possible formation of a bidentate hydrogen bond between Asp(16) 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 K (Ado), all the Asp(16) 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 Arg(131) and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg(131) residue as an effector-of product-mediated enzyme regulation is proposed.
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| Publisher |
PORTLAND PRESS LTDLONDONTHIRD FLOOR, EAGLE HOUSE, 16 PROCTER STREET, LONDON WC1V 6 NX, ENGLAND
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| Date |
2011-09-20T12:12:20Z
2011-09-20T12:12:20Z 2006 |
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| Type |
Article
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| Identifier |
BIOCHEMICAL JOURNAL
0264-6021 http://hdl.handle.net/123456789/14226 |
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| Language |
English
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