Aug 1, 1976

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

Journal of Bacteriology
W H TaylorP S Gilchrist

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 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.

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Mentioned in this Paper

Aspartic Acid, Magnesium-Potassium (2:1:2) Salt
Metalloproteins
Edetic Acid, Sodium Salt
Zinc
Clostridium
Clostridium Infections
Zinc Orotate
Orthophosphate
Cell-Free System
Hydrogen-Ion Concentration

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