Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.
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 [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine 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.
Bhaumik
D
D
Leishmania Group, Indian Institute of Chemical Biology, Calcutta, India.
Datta
A K
AK
D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008778:Methylthioinosine / Q000378:metabolism / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism*; D013379:Substrate Specificity; D014372:Tubercidin / Q000378:metabolism / Q000494:pharmacology
D008778:Methylthioinosine; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000248:Adenosine Kinase; D000241:Adenosine; D014372:Tubercidin