Purification and characterization of thymidylate synthetase from rat regenerating liver.
Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.
D000818:Animals; D002412:Cations / Q000494:pharmacology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008115:Liver Regeneration; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D002412:Cations; D013940:Thymidylate Synthase; D004229:Dithiothreitol