Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase

摘要: The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH bind at separate active sites separated by ∼60 A that are bridged four Fe4S4 centers. We have shown DPD undergoes reductive activation, taking up two [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate rate turnover not controlled protonation state general acid, cysteine 671. activation C671 variants delineated into phases particularly low values. Spectral deconvolution reaction reveals initial phase results in accumulation charge transfer absorption added binding difference spectrum for second reduction one flavins. X-ray crystal structure analysis C671S variant soaked slow substrate, thymine, a low-oxygen atmosphere resolved presumed activated form enzyme has FMN cofactor reduced. These data reveal arises proximity FAD bases ensuing flavin result rapid without reduced forms or suggest governed movement mobile structural feature carries residue.