Date of Award
10-16-2023
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Graham Moran
Abstract
Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil (5FU) as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the behavior of the enzyme had remained elusive. In this study we use transient state kinetic analysis methods to define the catalytic mechanism of DPD and elucidate a mode of enzyme inhibition by 5-ethynyluracil (5EU). Kinetic observations of anaerobic catalytic turnover determined the active form of DPD has the cofactor set of FAD-4(Fe4S4)-FMNH2. This two-electron reduced state is consistent with fewer mechanistic possibilities, and data suggests that the instigating and rate determining step in the catalytic cycle is reduction of the pyrimidine substrate that is followed by relatively rapid oxidation of NADPH at the FAD that, via the electron conduit of the 4(Fe4S4) centers, reinstates the FMNH2 cofactor for subsequent catalytic turnover. DPD inhibition by 5EU is shown to occur only in the presence of NADPH. When bound in the pyrimidine active site 5EU is shown to act as a DPD effector, inducing the enzyme to attempt flavin reduction using available NADPH. This process causes a protein loop that contains a general acid residue necessary for pyrimidine reduction, Cys671, to move within 4 Å of the bound 5EU. This distance is sufficient for thiol-yne click chemistry to occur between the C5 ethynyl group and the cysteine thiol, covalently attaching 5EU thereby inactivating DPD.
Recommended Citation
Forouzesh, Dariush, "Mammalian Dihydropyrimidine Dehydrogenase: Employing Transient State Kinetic Methods to Determine Catalytic and Inhibitory Mechanisms" (2023). Dissertations. 4042.
https://ecommons.luc.edu/luc_diss/4042
