Abstract
Thymidylate synthetase (EC 2.1.1.45) from rat regeneratign 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 suflate 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 μM and 65 μM, 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 K i value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent K i value of 23 μM.
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