The role of phosphate in the action of thymidine phosphorylase inhibitors: Implications for the catalytic mechanism

The inhibition of thymidine phosphorylase by AIFU was determined to follow uncompetitive kinetics with respect to inorganic phosphate, indicating that zwitterionic transition state analogs bind to the enzyme phosphate binary complex, consistent with an S N2-type catalytic mechanism. The design and synthesis of 5-fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU), a potent inhibitor of thymidine phosphorylase (TP) with K i-values of 11 nM (ecTP) and 17 nM (hTP), are described. Kinetic studies established that the type of inhibition of TP by AIFU is uncompetitive with respect to inorganic phosphate (or arsenate). The results obtained suggest that AIFU and other zwitterionic thymine analog inhibitors of TP act as transition state analogs, mimicking the anionic thymine leaving group, consistent with an S N2-type catalytic mechanism, and anchored by their protonated side chains to the enzyme-bound phosphate by electrostatic and H-bonding interactions.