Purification of properties of dihydroorotase, a zinc-containing metalloenzyme in Clostridium oroticum.
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 Clostridium 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 muM 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 Km 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 catalytic activity of the enzyme.
D000581:Amidohydrolases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002474:Cell-Free System; D003013:Clostridium / Q000201:enzymology* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000032:analysis; D008970:Molecular Weight; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D015032:Zinc / Q000032:analysis
D008667:Metalloproteins; D010618:Phenanthrolines; D010710:Phosphates; D001224:Aspartic Acid; D009963:Orotic Acid; D004492:Edetic Acid; D000581:Amidohydrolases; D015032:Zinc