Portland State University. Department of Biology
W. Herman Taylor
Term of Graduation
Date of Publication
Master of Science (M.S.) in Biology
Pyrimidines, Anaerobic bacteria, Enzymes -- Synthesis
1 online resource (v, 52,  pages)
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, CO2 and NH3 as reported by Liebenmm and Kornberg (7). Aspartate and CO2 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 O2, 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.
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Amy, Penny, "Enzyme Reactions Using Ureidosuccinate as a Substrate During Pyrimidine Biosynthesis and Degradation in Cl. oroticum" (1974). Dissertations and Theses. Paper 2153.