Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.