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.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-2688 |
| Date | 04 June 1973 |
| Creators | Balch, William Edward |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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