Stereoelectronic activation of methylenetetrahydrofolate by thymidylate synthase: resonance Raman spectroscopic evidence.

ABSTRACT

Resonance Raman (RR) spectra are reported for the ternary complex of Escherichia coli thymidylate synthase with the cofactor 5,10-methylenetetrahydrofolate (CH2-H4-folate) and the inhibitor 5-fluoro-2'-deoxyuridylate, excited at 337 or 356 nm, in resonance with perturbed absorption bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cofactor. For comparison, RR spectra were obtained with 260 nm excitation for PABA-Glu in various solvents, and for CH2H4-folate and H4-folate in aqueous solution. These reference spectra are assigned to modes of PABA-Glu in its benzenoid form. The ternary complex RR spectra are very different, however, and are assigned, with the aid of isotopic data, to the PABA-Glu in a predominantly quinoid form. Similar spectra were obtained for the ternary complexes of the E58Q and K48Q mutants, indicating that neither Glu58 nor Lys48 are essential for maintaining the quinoid structure, even though their side chains complement the dipolar charge distribution of the quinoid form of PABA-Glu. Since these are the only charged residues in the PABA-Glu vicinity, electrostatic stabilization is not essential to maintenance of the quinoid structure. It is proposed that quinoid formation results from steric forces, probably resulting from the protein conformation change known to accompany cofactor binding, which enforce coplanarity of the PABA-Glu ring and substituents. This stereoelectronic change activates the cofactor by opening the methylene bridge. A second RR spectrum of the ternary complex, previously proposed to reflect an alternate structure, is shown to result instead from irreversible formation of a laser-induced photoproduct.