Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani

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 show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in K_m (Ado) with a 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 Asp¹⁶ in the maintenance of steric complementarity of the binding pocket. Use of 2'or 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 (ΔΔG^B) contribution of the former was 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 K_m (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.