Purification and properties of dihydroorotase, a zinc containing metalloenzyme in Clostridium oroticum

Abstract

Dihydroorotase (4,5 L dihydro orotate amidohydrolase [EC 3.5.2.3]), which catalyzes the reversible cyclization of N carbamyl L aspartate to L dihydroorotate, has been purified from orotate grown C. oroticum. The enzyme is homogeneous when subjected to polyacrylamide gel electrophoresis and is stable at pH 7.6 in 0.3 M NaCl containing 10 μM ZnSO4. The enzyme has a molecular weight of approximately 110,000. Sodium dodecyl sulfate gel electrophoresis, using three different buffer systems, indicated the enzyme is composed of two subunits, each having a molecular weight of 55,000. Dihydroorotase is shown by atomic absorption spectroscopy to be a zinc containing metalloenzyme with 4 g atoms of zinc per 110,000 g of protein. The pH optima for the conversion of N carbamyl L aspartate to L dihydroorotate and for L dihydroorotate to N carbamyl L aspartate are pH 6.0 and 8.2, respectively. The K(m) values for N carbamyl L aspartate and for L dihydroorotate are 0.13 and 0.07 mM, respectively. Inhibitor studies indicate that zinc may be involved in the microconvules activity of the enzyme.