Assay ofEscherichia coliDihydroorotase with Enantiomeric Substrate: Practical Preparation of Carbamyll-Aspartate and High-Performance Liquid Chromatography Analysis of Catalysis Product

Abstract A reasonably facile and effective procedure is described for preparing the optically active substrate of dihydroorotase (EC 3.5.2.3), N -carbamyl- l -aspartate (L-CA), which is not commercially available. Compared to the previously described methods, this procedure is not plagued by side reactions, and the L-CA is obtained in a solid form as the Mg 2+ /K + mixed salt. In that salt form, the L-CA can be prepared in large quantity, and it is easier to handle and stable upon storage. L-CA can be separated from aspartate and its cyclic derivatives, dihydroorotate and hydantoin, by HPLC using a strong anion-exchange column. This HPLC method, which is used to check the purity of the synthesized carbamyl aspartate, is also effective for monitoring the enzymatic reaction.