5-Fluoro-2′-deoxycytidine 5′-monophosphate is a mechanism-based inhibitor of thymidylate synthase

Abstract

Thymidylate synthase (TS) is inhibited by 5-fluoro-2′-deoxycytidine 5′-monophosphate (FdCMP). From initial velocity measurements, the apparent K i for the binary FdCMP-enzyme complex was about 20 μM. In the presence of 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2H 4folate), FdCMP causes a time-dependent inactivation of the enzyme and formation of a TS-FdCMP-CH 2H 4folate complex. The ternary complex contains one mol of inhibitor per monomer of enzyme, and can be readily isolated on nitrocellulose filters. Dissociation of the ternary complex is quite slow ( t l 2 ≈ 16 h ), and yields unchanged FdCMP. As with the corresponding complex formed with 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP), the TS-FdCMP-CH 2H 4folate complex shows a differential absorbance maximum at 326 nm, and is stable to SDS-PAGE. Taken together, these results indicated that FdCMP is a slow, tight binding inhibitor of TS and has a mechanism of inhibition similar to that of FdUMP.