Isolation and characterization of adenosine kinase from Leishmania donovani.
Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified 3250-fold from Leishmania donovani promastigotes using ion-exchange, gel filtration, and affinity chromatography techniques. Both native and sodium dodecyl sulfate-gel electrophoresis of the enzyme revealed a single polypeptide of around 38,000 molecular weight. Biophysical and biochemical analyses of the enzyme reveal unique characteristics different from those of adenosine kinases from other eukaryotic sources. The isoelectric pH of the enzyme is 8.8. In native acrylamide gels the enzyme moves with an RF of about 0.62. The enzyme displays a maximum activity at pH between 7.5 and 8.5 and is dependent upon an optimum ATP/Mg2+ ratio. ATP at high concentration inhibits the reaction. Adenosine and Mg2+ are not inhibitory. EDTA completely knocks off the activity. Enzyme activity is dependent upon the presence of active thiol group(s) at or near the active center. Under a defined set of conditions the enzyme exhibited an apparent Km for adenosine and ATP of 33 and 50 microM, respectively. Of the nucleoside triphosphates tested ATP and GTP were the most effective phosphate donors. Marginal inhibition of activity was detected with other nucleosides as competitors. However, adenosine analogs, such as 7-deaza-adenosine (tubercidin) and 6-methylmercaptopurine riboside at very low concentrations, were found to be excellent inhibitors and substrates as well. S-Adenosylhomocysteine does not inhibit the reaction even at very high concentration.
D000248:Adenosine Kinase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D006801:Humans; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000302:isolation & purification; D008970:Molecular Weight; D009705:Nucleosides / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D013045:Species Specificity; D013379:Substrate Specificity
D009705:Nucleosides; D009711:Nucleotides; D010770:Phosphotransferases; D000248:Adenosine Kinase