Properties of Thymidylate Synthetase from Dichloromethotrexate-resistant Lactobacillus casei

Extracts of dichloromethotrexate-resistant Lactobacillis casei provide a rich source of stable thymidylate synthetase, showing 100 times the activity found in extracts of the sensitive strain. The enzyme was purified 17-fold from extracts of resistant cells. It has a molecular weight of 67,000 and is activated by 2-mercaptoethanol. The rate of the reaction as determined by loss of tritium from deoxyuridine monophosphate-5-3H was 84% of the rate observed spectrophotometrically. The enzyme from dichloromethotrexate-resistant L. casei differs from Escherichia coli thymidylate synthetase, in that it will utilize tetrahydrohomofolate as a cofactor in place of tetrahydrofolate. The Km values for tetrahydrofolate and tetrahydrohomofolate are 4.5 x 10-5 m and 7.2 x 10-6 m, respectively, showing that tetrahydrohomofolate is bound to the enzyme more tightly than tetrahydrofolate, although the Vmax obtained with tetrahydrohomofolate is only 6% of that obtained with tetrahydrofolate. The Ki of tetrahydrohomofolate of 4.6 x 10-6 m is close to the Km for that compound. 10-Methyltetrahydrofolate and 11-methyltetrahydrohomofolate were inactive as cofactors. They inhibited the reaction 50% at 4 x 10-6 m and 3 x 10-5 m, respectively. Tetrahydrohomopteroate was neither a substrate nor an inhibitor at 3 x 10-4 m. Methotrexate-resistant strains of L. casei also showed high levels of thymidylate synthetase activity.