Dihydroorotate dehydrogenase. I. General properties.

Abstract An improved method is described for the preparation of crystalline dihydroorotate dehydrogenase which is electrophoretically homogeneous. The enzyme exhibits an s20,w value of 7.45 S at infinite dilution, and its molecular weight is 115,000 ± 7,000. It contains 2 moles of FAD, 2 moles of FMN, 4 g atoms of iron, and 4 moles of acid-labile sulfide per mole of enzyme. Apparent Km values were determined for DPNH, orotate, and dihydroorotate. Orotate and oxidized enzyme yield a complex characterized by its absorption spectrum. Thermodynamic parameters for the dissociation of this complex are presented. Substrate specificity was studied with a series of pyrimidine derivatives. The ring hydroxyl and carboxyl substituents are essential for binding and oxidation of reduced enzyme. Halogen substitution at Position 5 increases both Km and Vmax. Reduction of the enzyme by reducing substrates is evidenced by bleaching at 450 mµ and the appearance of long wave length absorption. Reduction by DPNH proceeds rapidly; reduction by dihydroorotate is slow and proceeds to about one-half the extent found with DPNH. However, reduction of the enzyme by dihydroorotate in the presence of DPN is as complete, and perhaps as rapid, as that by DPNH. A large Cotton effect, observed in the visible region of the spectrum, was markedly altered by dithionite or reducing substrate and was shifted to lower wave lengths by 3.5 m urea. A flavin-free iron-protein was obtained which exhibited an absorption spectrum similar to that of iron-proteins from other iron-flavoproteins and to that of spinach ferredoxin. Three techniques were used to demonstrate the formation of an oxygen free radical when enzyme, reduced by substrate, is reoxidized by oxygen. Treatment with cysteine is required for reactions involving orotate or dihydroorotate, but not for DPNH oxidase activity. Similarly, treatment with mercuribenzoate destroyed orotate reductase and dihydroorotate oxidase activity while inhibiting DPNH oxidase activity only slightly. A tentative scheme for the internal electron transport system of this enzyme is presented.