ANGIOSTRONGYLUS-CANTONENSIS - CHARACTERIZATION OF THYMIDYLATE SYNTHETASE

Thymidylate synthetase (TS) is the only enzyme that catalyzes the form ation of thymine nucleotides in Angiostrongylus cantonensis. A fractio n enriched in TS was obtained from the gravid nematode by gel filtrati on and affinity chromatography using methotrexate-agarose. TS, which w as well separated from dihydrofolate reductase, has a relative molecul ar mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band w as shown to be TS by comparing the electrophoretic mobility with an en zyme preparation bound with [6-H-3]S-fluoro-2'-deoxyuridine 5'-monopho sphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition pattern s revealed that the TS reaction undergoes a sequential mechanism in wh ich 2'-deoxyuridine 5'-monophosphate (dUMP) is the first substrate add ed to the active site and thymidine 5'-monophosphate is the last produ ct released. The apparent Km values for dUMP and 5, 10-methylenetetrah ydrofolate are 10 and 185 mu M, respectively. FdUMP and trimethoprim i nhibited the parasite TS competitively with dUMP and the K-i values of 23.5 nM and 852 mu M, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5, 10-methylenetetrafolate, 1 mM methotrex ate inhibited the activity by 74%. (C) 1994 Academic Press, Inc.