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

Abstract

Abstract—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.