Trapping Substrate in a Strained Conformation: Crystal Structures of Thymidylate Synthases. William R. Montfort, Bryan Arendall, David C. Hyatt, Department of Biochemistry, University of Arizona, Tucson, AZ 85721

Two crystal structures of E. coli thymidylate synthase (TS) have been determined with cofactor methylenetetrahydrofolate (CH₂THF) trapped in a disfavored conformation that is apparently required for catalysis. The first structure is to 1.9 Å nominal resolution and contains wild-type TS, CH₂THF, and the mechanism-based inhibitor 5-NO₂-dUMP (NO₂dUMP). The second is to 2.0 Å nominal resolution and contains the nearly inactive mutant C146A, CH₂THF, and substrate dUMP, and thus provides a rare view of a true substrate complex. TS catalyzes the conversion of dUMP to dTMP by transferring a methylene group from a 5-membered ring in CH₂THF to dUMP. Previous structures of TS and CH₂THF have displayed cofactor after ring opening, in a conformation thought to be unobtainable before ring opening, due to strain in the ring. In the present structures, cofactor is in the "bent" conformation previously seen, but with an intact ring. TS undergoes a substantial conformational change during catalysis, and the new structures suggest TS uses its "open" conformation to bind dUMP and the solution conformer of CH₂THF, followed by closure of the active site and stabilization of the higher-energy bent conformer. Catalysis of ring opening and methylene transfer follow. These results will be discussed in the context of our recent structures of 5-fluoro-dUMP (FdUMP), CH₂THF, and TS [1], in which we obtained a slightly different ligand arrangement from that previously observed for this complex [2], and which led us to propose an alternate mechanism for catalysis by TS. In this proposed mechanism, we suggest that variability in the covalent bond between Cys 146 of TS and dUMP, which has been observed to varying degree in structures of TS complexes, is a necessary component of catalysis.

[1] D.C. Hyatt, F. Maley, W. R. Montfort (1997), Biochemistry, in press.

[2] D. A. Matthews et al. (1990), J. Mol. Biol. 214:937-948.