Catalysis in OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10) is coupled to the unstructured-to-structured transition of a 10-residue peptide loop. OMP synthase from yeast is a small, homodimeric (49 kDa) and highly stable domain-swapped enzyme that catalyzes the formation of the UMP precursor orotidine 5'-monophosphate (OMP) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA). Inter-subunit communication has been proposed to result in half-of-the-sites cooperativity of binding [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342]. High-resolution X-ray structures of the highly homologous Salmonella typhimurium and E. coli enzymes show that major and apparently essential movement of protein (mobile peptide loops and entire core) and substrates accompany catalysis and can result in an occupied enzyme showing asymmetry.A novel unproductive OA analog, citrazinic acid (CA), allows structural exploration of stable Michaelis-like complexes by NMR. Here, we report the backbone 1H-13C-15N NMR assignments of the yeast enzyme in unliganded, OMP and MgPRPP·CA forms. Equilibrium binding of PRPP in the presence of CA shows two independent binding sites present in equal concentrations, but with KD values 19-fold lower and 3-fold higher than the single for PRPP of 6.3 ± 0.6 μM to two sites in the native dimer. Binding and potential structural asymmetry was further investigated by isothermal titration calorimetry and ligand titration by NMR. The implications for cooperativity and the origin of the domain swapped structure are discussed.