PHOTOSENSITIZED REDUCTION OF L-BIOPTERIN IN THE ACTIVE TERNARY COMPLEX OF DIHYDROFOLATE-REDUCTASE

Photosensitized L-biopterin induces the transfer of a hydrogen atom fr om the dihydronicotinamide moiety of NADPH to the biopterin ring. Sens itization occurs through the triplet state of both the lactim and lact am tautomers of L-biopterin. Quenching kinetic analysis to measure the bimolecular rate constant demonstrated a greater reactivity for the l actim tautomer. Recombinant human dihydrofolate reductase enzyme, for which these molecules are substrate and cofactor, enhanced the rate co nstant of the photosensitized H-. transfer to the apparent lactim taut omer by eight times to 1.6 X 10(10) M(-1) s(-1) When NADP(+) replaced NADPH at the active site, no enhanced photoreduction of biopterin was observed, implying that the hydrogen atom comes from the reduced nicot inamide group and not as a result of protein conformational changes. T his reduction at the active site represents a photoinitiated H-. trans fer in protein between substrate and cofactor.