ABSTRACT
Resonance Raman (RR) spectra are reported for the binary complex of Escherichia coli thymidylate synthase (TS) with the substrate analog inhibitor 5-nitrodeoxyuridylate (NDU). The TS/NDU binary complex RR spectrum shows many similarities to the RR spectra of thiol adducts of NDU or of 5-nitro-1-methyluracil formed in solution, providing strong evidence in support of the formation of a covalent link between Cys146 of TS and C6 of NDU. Spectral differences between the model compounds and the binary complex reflect the consequences of fixing the conformations of the uracil and ribose rings at the enzyme active site. The RR spectra of the ternary complexes of TS/NDU with either tetrahydrofolate (H4-folate) or the cofactor 5,10-methylenetetrahydrofolate (CH2H4-folate) show that a covalent link is not formed between C11 of CH2H4-folate and C5 of NDU. Neither does the methylene bridge of CH2H4-folate remain intact in the ternary complex; either CH2H4-folate is present as the N5 iminium cation species or the methylene group is lost as formaldehyde. A shift in the NO2 symmetric stretching frequency in the ternary complex indicates expulsion of water molecules from the region of the NO2 group by the cofactor.
