Angiostrongylus cantonensis: Characterization of Thymidylate Synthetase

Thymidylate synthetase (TS) is the only enzyme that catalyzes the formation of thymine nucleotides in Angiostrongylus cantonensis. A fraction enriched in TS was obtained from the gravid nematode by gel filtration and affinity chromatography using methotrexate-agarose. TS, which was well separated from dihydrofolate reductase, has a relative molecular mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band was shown to be TS by comparing the electrophoretic mobility with an enzyme preparation bound with [6-3H]S-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition patterns revealed that the TS reaction undergoes a sequential mechanism in which 2′-deoxyuridine 5′-monophosphate (dUMP) is the first substrate added to the active site and thymidine 5′-monophosphate is the last product released. The apparent Km values for dUMP and 5, 10-methylenetetrahydrofolate are 10 and 185 μM, respectively. FdUMP and trimethoprim inhibited the parasite TS competitively with dUMP and the Ki values of 23.5 nM and 852 μM, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5, 10-methylenetetrafolate, 1 mM methotrexate inhibited the activity by 74%.