Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani

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Abstract

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [γ-32P]ATP resulted in binding of adenosine to free enzyme. Tubercidin (7-deazaadenosine) and 6-methylmercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. ‘Substrate efficiency (Vmax/Km)’ and ‘turnover numbers (Kcat)’ of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

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Cited by (22)

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    However, beyond this ribose-induced closure of its small and large domains, only small tertiary structural changes are found among the apo, ribose and ribose:ADP-bound enzyme forms, and many aspects of its catalytic mechanism are still unclear (Sigrell et al., 1999). Similarly, the structure of the human AK:adenosine complex, although providing some insight into substrate binding, left many questions about its catalytic mechanism unanswered; the pertinent biochemical data regarding the enzymatic mechanism are equivocal and controversial, and thus, this structure could not resolve all issues (Henderson et al., 1972; Palella et al., 1980; Rotllan et al., 1985; Hawkins et al., 1987; Bhaumik et al., 1988). In order to understand better the structural and mechanistic basis for phosphate transfer by this class of carbohydrate kinases, we determined the structures of the T. gondii apo AK to 2.55 Å resolution, the AK:adenosine complex to 2.50 Å resolution, and the AK:adenosine:AMP-PCP (a non-hydrolysable ATP analog) ternary complex to 1.71 Å resolution.

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    However, beyond this ribose-induced closure of its small and large domains, only small tertiary structural changes are found among the apo, ribose and ribose:ADP-bound enzyme forms, and many aspects of its catalytic mechanism are still unclear (Sigrell et al., 1999). Similarly, the structure of the human AK:adenosine complex, although providing some insight into substrate binding, left many questions about its catalytic mechanism unanswered; the pertinent biochemical data regarding the enzymatic mechanism are equivocal and controversial, and thus, this structure could not resolve all issues (Henderson et al., 1972; Palella et al., 1980; Rotllan et al., 1985; Hawkins et al., 1987; Bhaumik et al., 1988). In order to understand better the structural and mechanistic basis for phosphate transfer by this class of carbohydrate kinases, we determined the structures of the T. gondii apo AK to 2.55 Å resolution, the AK:adenosine complex to 2.50 Å resolution, and the AK:adenosine:AMP-PCP (a non-hydrolysable ATP analog) ternary complex to 1.71 Å resolution.

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