Research done in collaboration with Sondra Berger, USC-Basic Pharmaceutical Sciences. Thymidylate Synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridylate (dUMP) by CH2H4folate to produce Thymidylate (TMP) and H2folate. Because of its role in DNA replication, the enzyme has been a target of chemotherapeutic agents. By inhibiting this synthesis, the cell cycle is inhibited and cell death occurs by thymidineless stress. This equates to a cancerous cell dying from TS inhibition. In an inactive conformation of hTS, the catalytic cysteine is no longer positioned in the ligand-binding pocket. The nucleophilic sulfhydryl group of cysteine is probably the most reactive functional group in proteins. Both TS catalytic activity and TS ternary complex formation with FdUMP and CH2H4folate require that the conserved cysteine sulfhydryl positioned in the enzyme active site is fully reduced. Previous studies indicate that Ser216 of hTS is involved in dUMP binding and has effects on catalysis. The reaction rates of cysteine residues with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid; DTNB) were slower for Ala216 TS than for wild-type TS. We will study the rates of modification of cysteine residues in wild-type and mutant hTS. We will also utilize Capillary Electrophoresis to examine the extent of anion formation of the sulfhydryl group. Thermal denaturation studies will determine the extent of disulfide bond formation in mutant hTS affecting the stability of the protein. These results will provide evidence that Ser216 is playing a role in activation of the active site cysteine in human TS.