Thymidylate synthase (TS) is a homodimeric enzyme that catalyses the reductive methylation of deoxyuridinemonophosphate (dUMP) to thymidinemonophosphate (dTMP) using methylenetetrahydrofolate as both a one carbon donor and a reducing equivalent. The enzyme is an anti-cancer drug target and is investigated for its potential use as a drug target in parasitic and viral diseases as well. The long-standing interest of the laboratory has been to correlate the importance of residues at the active site of the enzyme from TS to its catalytic function. Mutants of key residues thought to be important for function are made and characterized by steady state kinetic analysis and their structure analyzed by X-ray crystallography. Using a panel of C terminal mutants from L. casei, we have been able to show that the ternary complex between substrate, cofactor and enzyme at the structurally identical active sites are non-equivalent in their reactivities. Mutations of residues elsewhere in the active site are being pursued to delineate the late events in the catalytic pathway of this very important enzyme in nucleotide biosynthesis.
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