Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia.
Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.
D002846:Chromatography, Affinity; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009703:Nucleoside-Phosphate Kinase / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D013938:Thymidine Monophosphate
D013938:Thymidine Monophosphate; D008345:Manganese; D010770:Phosphotransferases; D009703:Nucleoside-Phosphate Kinase; D008274:Magnesium