Human thymidylate synthetase derived from blast cells of patients with acture myelocytic leukemia. Purification and chracterization.
Thymidylate synthetase (EC 2.1.1.B.) from blast cells of patients with acute myelocytic leukemia has been purified more than 1470-fold by affinity column chromatography. Methotrexate was the affinity ligand. dUMP was found to be a necessary additive for retention of the enzyme by the affinity column. Disc electrophoresis and sucrose density gradient centrifugation revealed a single enzyme species with a molecular weight of 76,000. The enzyme exhibits a temperature-dependent conformational change with activation energies of 5.3 +/- 0.4 and 17.3 +/- 1.9 kcal/mol, respectively, above and below a transitional temperature of 35 degrees. This conformational change is reflected in the binding affinity of dUMP but not of 5,10-methylenetetrahydrofolate. The enzyme displays a broad pH maximum in the range of pH 7.4 to 8.8. The Michaelis constants for dUMP and (+/--L-5,10-methylenetetrahydrofolate are 1.8 +/- 0.2 and 31 +/- 8.3 micrometer, respectively. Initial velocity and product inhibition studies reveal the enzymic mechanism to be ordered sequential. dUMP binds before 5,10-methylenetetrahydrofolate and dihydrofolate is released before TMP. 5-Fluoro-2'-deoxy-5'-uridylate (FdUMP) behaves as in irreversible inhibitor with a Ki of 1.68 +/- 0.45 nM. The enzyme has a turnover number of 6 min-1 per FdUMP binding site. Methotrexate inhibits noncompetitively with respect to dUMP and binds tighter to the enzyme in the presence of dUMP. Methotrexate antagonizes inactivation of the enzyme by FdUMP.
D001755:Blastocyst / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000302:isolation & purification / Q000378:metabolism*
D008780:Methyltransferases; D013940:Thymidylate Synthase; D008727:Methotrexate