Figure 6. Figure 6, see also Figures S5 and S6: Uric acid is a direct inhibitor of UMPS.
(A) Schematic depicting the reactions catalyzed by each domain of bifunctional UMP synthase (UMPS). OPRT: orotate phosphoribosyltransferase. ODC: OMP decarboxylase.
(B) Schematic depicting competitive inhibition of the ODC domain of UMPS by allopurinol ribonucleotide (top) and 6-aza-UMP (bottom). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of allopurinol to its ribonucleotide derivative, and uridine-cytidine kinase (UCK) catalyzes that of 6-azauridine to 6-aza-UMP.
(C) Quantification of OMP (left) and UMP (right) following incubation of recombinant UMPS (WT or the Y37A, R155A mutant) with its substrates orotate and PRPP at the indicated concentrations (mean ± SD, n = 3). WT: wild-type.
(D) Relative abundances of OMP (left) and UMP (right) following addition of increasing concentrations of 6-aza-UMP or vehicle to the UMPS activity assay (mean ± SD, n = 3).
(E) Relative abundances of OMP (left) and UMP (middle) following addition of increasing concentrations of uric acid or vehicle to the UMPS activity assay (mean ± SD, n = 3). Schematic depicting competitive inhibition of the ODC domain of UMPS by uric acid (right).
(F) Relative abundances of OMP (left) and UMP (right) following addition of 9-methyluric acid, allantoin, allopurinol, 6-azauridine, uric acid, or vehicle to the UMPS activity assay (mean ± SD, n = 3).