Cells of Clostridium oroticum, an anaerobic bacterium, were grown on orotate as a carbon and energy source. Ureidosuccinase, an inducible enzyme in the pathway for pyrimidine degradation has been shown to convert ureidosuccinate to aspartate, C02 and NH3 as reported by Liebenmm and Kornberg (7). Aspartate and C02 were formed in approximately a 1: 1 ratio from ureidosuccinase activity. Ureidosuccinase was found to be a Mn+2 requiring enzyme with a pH optimum of approximately pH 6.5. Enzyme activity is labile to 02, temperature, pH, dilution and high ionic strength. The optimum conditions for storage of ureidosuccinase were found to be mixtures which contained Mn+2, PO4-3 and ureidosuccinate (the substrate of the enzyme) at -20°C in argon gassed serum vials. Hydantoinase, an enzyme which converts ureidosuccinate to 5'-carboxymethylhydantoin in a ratio of two 5'-carboxymethylhydantoin to one ureidosuccinate, was present in cells grown on orotate. Hydantoinase was able to convert both the D and the L isomers of ureidosuccinate to carboxymethylhydantoin. High levels of a Mg+2 dependent carbamyl phosphate kinase were found in extracts from cells grown on orotate. The activity was dependent upon addition of ADP or AMP to the reaction mixture. The pH optimum of the carbamyl phosphate kinase was approximately pH 6.5. Cells grown on orotate contained high levels of an induced system for the degradation of pyrimidines whereas cells grown on glucose contained a constitutive level of enzymes for pyrimidine biosyntheses. Low levels of ureidosuccinase, carbamyl phosphate kinase and hydantoinase were found in glucose grown cells. Cells grown on glucose showed a high level of a Mn+2 dependent aspartate transcarbamylase (the analogous enzyme to ureidosuccinase which is operative during pyrimidine biosynthesis) which forms ureidosuccinate from aspartate and carbamyl phosphate. A consideration of the energy and reducing power supply and demand was made for cells grown both on glucose and orotate as carbon and energy source.

Dihydroorotase (4,5-L-dihydro-orotate amidohydrolase, EC 3.5.2.3) which catalyzes the reversible cyclization of N-carbamyl-1-aspartate toL-dihydro-orotate has been purified from orotate-grown Clostridium oroticum by a combination of streptomycin sulfate fractionation, DEAE-Sephadex chromatography, and hydroxylapatite chromatograpy. The enzyme has been shown to be omogeneous when subjected to polyacrylamide gel electrophoresis. Thin-layer gel chromatography with Sephadex G-200 indicated the enzyme to have a molecular weight of 110,000 ± 10,000. Sodium dodecyl sulfate gel electrophoresis using two different buffer systems indicate the enzyme to be composed of two identical subunits with a molecular weight of 56,000 ± 5300. Dihydroorotase has been shown to be a zinc-containing metalloenzyme by atomic absorption spectroscopy with two g atoms of zinc per 56,000 g of protein. The pH optima for the conversion of N-carbamyl-L-aspartate to L-dihydroorotate and L-dihydroorotate to N-carbamyl-L-aspartate have been determined to be at pH 6.0 and pH 8.2 respectively. The binding constant of the enzyme for each substrate has been investigated with values of 0 .13 mM for N--carbamyl-L--aspartate and .07 mM for L-dihydroorotate.