Assay of Escherichia coli dihydroorotase with enantiomeric substrate: practical preparation of carbamyl L-aspartate and high-performance liquid chromatography analysis of catalysis product.
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 Mg2+/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.
Daniel
R
R
Laboratoire de Recherches sur les Macromolécules, URA 502 CNRS, Université Paris 13, avenue J.-B. Clément, Villetaneuse, 93430, France.
Kokel
B
B
Caminade
E
E
Martel
A
A
Le Goffic
F
F
D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004080:Dihydroorotase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D013237:Stereoisomerism
D001224:Aspartic Acid; D004080:Dihydroorotase; D008274:Magnesium; C006893:ureidosuccinic acid; D011188:Potassium