Substrate Recognition and Activation by Two Flavoenzymes Involved in Pyrimidine Metabolism:Flavin-Dependent Thymidylate Synthase and tRNA-Dihydrouridine Synthase.

[摘要] Pyrimidines are essential components of nucleic acids. In biology, they undergo a number of redox reactions catalyzed by flavin-dependent enzymes. This thesis investigates the mechanism of substrate recognition and activation of two flavin-dependent enzymes with pyrimidine-containing substrates: flavin-dependent thymidylate synthase (FDTS) and tRNA-dihydrouridine synthase (DUS). FDTS catalyzes the reductive methylation of the uracil moiety of 2’-deoxyuridine-5’-monophosphate (dUMP) into thymine, whereas DUS reduces specific uracils in tRNA to dihydrouracil. NMR data indicate that FDTS ionizes N3 of the uracil in dUMP using an active-site arginine, which is proposed to initiate catalysis by enhancing the nucleophilicity of C5 of the uracil. Biochemical data on dUMP analogs suggests that the phosphate of dUMP acts as the base that removes the proton from C5 of dUMP during the FDTS-catalyzed reaction. Notably, both ionization of N3 and acid-base catalysis by the phosphate of dUMP are not implicated in the mechanism used by human thymidylate synthase.Several equilibrium and kinetic methods were used to study the mechanism of deoxynucleotide recognition by Thermotoga maritima FDTS. FDTS binds deoxynucleotides with ~200-fold weaker affinity when the flavin is reduced relative to when it is oxidized, and the differences in affinity are largely due to differences in the dissociation rate constant. There is also a temperature-dependent effect on the mechanism by which FDTS binds deoxynucleotides – below 45°C the FDTS homotetramer behaves as a dimer-of-dimers with deoxynucleotide binding while at temperatures above 45°C the four subunits of FDTS bind deoxynucleotides identically.tRNAs contain a number of nucleobase and ribose modifications – including dihydrouracil – at different positions of the tRNA. Previous work has shown that yeast tRNALeu-CAA reacts poorly with yeast DUS2 unless it contains other modifications, suggesting that tRNA modifications are ordered. The reactivity of yeast DUS2 with other unmodified tRNAs was investigated; unmodified yeast tRNAAsp, tRNAAla, and tRNAHis2 all reacted rapidly with yeast DUS2, indicating that not all tRNAs require prior modifications to react rapidly with yeast DUS2. Native gel electrophoresis showed that unmodified tRNALeu-CAA misfolds, explaining its poor reactivity with yeast DUS2.

[发布机构] University of Michigan

[效力级别] Flavin

[关键词] Mechanistic enzymology;Flavin;Pyrimidine;Biological Chemistry;Science;Chemical Biology