STEREOELECTRONIC ACTIVATION OF METHYLENETETRAHYDROFOLATE BY THYMIDYLATE SYNTHASE - RESONANCE RAMAN-SPECTROSCOPIC EVIDENCE

Resonance Raman (RR) spectra are reported for the ternary complex of E scherichia coli thymidylate synthase with the cofactor 5,10-methylenet etrahydrofolate (CH2-H-4-folate) and the inhibitor 5-fluoro-2'-deoxyur idylate, excited at 337 or 356 nm, in resonance with perturbed absorpt ion bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cof actor. For comparison, RR spectra were obtained with 260 nm excitation for PABA-Glu in various solvents, and for CH2H4-folate and H-4-folate in aqueous solution. These reference spectra are assigned to modes of PABA-Glu in its benzenoid form. The ternary complex RR spectra are ve ry different, however, and are assigned, with the aid of isotopic data , to the PABA-Glu in a predominantly quinoid form. Similar spectra wer e obtained for the ternary complexes of the E58Q and K48Q mutants, ind icating that neither Glu58 nor Lys48 are essential for maintaining the quinoid structure, even though their side chains complement the dipol ar charge distribution of the quinoid form of PABA-Glu. Since these ar e the only charged residues in the PABA-Glu vicinity, electrostatic st abilization is not essential to maintenance of the quinoid structure. It is proposed that quinoid formation results from steric forces, prob ably resulting from the protein conformation change known to accompany cofactor binding, which enforce coplanarity of the PBBA-Glu ring and substituents. This stereoelectronic change activates the cofactor by o pening the methylene bridge. A second RR spectrum of the ternary compl ex, previously proposed to reflect an alternate structure, is shown to result instead from irreversible formation of a laser-induced photopr oduct.