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

— Photosensitized L-biopterin induces the transfer of a hydrogen atom from the dihydronico-tinamide moiety of NADPH to the biopterin ring. Sensitization occurs through the triplet state of both the lactim and lactam tautomers of L-biopterin. Quenching kinetic analysis to measure the bimolecular rate constant demonstrated a greater reactivity for the lactim tautomer. Recombinant human dihydro-folate reductase enzyme, for which these molecules are substrate and cofactor, enhanced the rate constant of the photosensitized H• transfer to the apparent lactim tautomer by eight times to 1.6 × 1010M-1s-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 nicotinamide group and not as a result of protein conformational changes. This reduction at the active site represents a photoinitiated H• transfer in protein between substrate and cofactor.