The Purification and Properties of Thymidylate Synthetase from Chick Embryo Extracts

Thymidylate synthetase was purified about 600- to 800-fold from 7-day chick embryo and was found to methylate uridine 5'-phosphate at 40% the rate of deoxyuridine 5'-phosphate. The pH optima and salt effects on the two reactions differed, but most of the evidence indicated that the same enzyme was responsible for both activities. The Km for deoxyuridine 5'-phosphate was 102- to 103-fold lower than that for uridine 5'-phosphate, depending on the assay employed. Chromatography on Sephadex G-200 and sucrose density gradient centrifugation revealed the molecular weight of the synthetase to be about 58,000. Phosphatases were found during the purification that hydrolyzed the pyrimidine ribonucleotides faster than the deoxyribonucleotides. The synthetase was protected against heat inactivation by deoxyuridine 5'-phosphate and, to a lesser extent, by 5,10-methylene tetrahydrofolate, but not by uridine 5'-phosphate. The activation energy for both deoxyuridine 5'-phosphate and uridine 5'-phosphate was 12 kcal per mole. Inhibition by 5-fluorodeoxyuridine 5'-phosphate appeared to be competitive with respect to deoxyuridine 5'-phosphate, but by incubating the enzyme with inhibitor prior to the addition of substrate, noncompetitive inhibition was obtained. Similar results were observed with 4-N-hydroxydeoxycytidine 5'-phosphate. Double reciprocal plots with fixed and variable substrates revealed that neither deoxyuridine 5'-phosphate nor 5,10-methylene tetrahydrofolate affected the Km of the other, an observation indicative of a random addition mechanism. However, product inhibition by thymidine 5'-phosphate was asymmetric since it was competitive with respect to deoxyuridine 5'-phosphate and noncompetitive with respect to 5,10-methylene tetrahydrofolate. The mechanism by which 1 of the hydrogen atoms of the thymine methyl group is derived was investigated with tetrahydrofolate labeled with tritium in positions 6 and 7 of the pteridine ring. The transfer was found to occur, without dilution, from position 6 only.