Crystal structure of a deletion mutant of human thymidylate synthase Delta(7-29) and its ternary complex with Tomudex and dUMP

The crystal structures of a deletion mutant of human thymidylate synthase ( TS) and its ternary complex with dUMP and Tomudex have been determined at 2 .0 Angstrom and 2.5 Angstrom resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type e nzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ter nary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Angstrom away f rom the catalytic cysteine in the open conformation. As a result, the coval ent bond between the catalytic cysteine sulfhydryl and the base of dUMP, wh ich is the first step in the reaction mechanism of TS and is observed in al l ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the prote in that are caused by differences in the environment of the glutamyl tail o f the Tomudex molecule. Despite the absence of the closed conformation, Tom udex inhibits human TS ten-fold more strongly than E. coli TS. These result s suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by elimina ting this as a condition for lead compounds.