Crystalline Dihydroorotic Dehydrogenase

Abstract

Evidence has previously been presented that dihydroorotic dehydrogenase from Zymobacterium oroticum (1) is a flavoprotein(2).The present paper describes a procedure for crystallizing the enzyme as a globulin.Data are also presented to show that the protein crystals contain about one atom of iron per molecule of flavin, and that the flavin consists of both flavin mononucleotide and flavin adenine dinucleotide, present in equimolar proportions.The bleaching of the flavin by the substrates and some other properties of the enzyme will also be described.EXPERIMENTAL Materials-Z.oroticum was the same as that previously used(2).Orotic acid and DPNH were obtained from Sigma Chemical Company.Protamine sulfate, lipoic acid (dl-thioctic acid), and 5-methyl erotic acid were obtained from Nutritional Biochemicals Corporation.Glass beads ("Superbrite" No. 100, diameter 0.2 mm) were obtained from the Minnesota Mining and ManufacturingCompany.Other materials were the same as previously described (2).Methods-Flavins were determined by the method of Bessey et al. (3) and Burch (4).In addition, FAD was determined enzymatically with the apoenzyme of n-amino acid oxidase (3, 5), and FMNl was estimated in an analogous mamer with the apoenzyme of pneumococcal lactic oxidase (6).FAD and FMN were also determined chromatographically as described in the following paper (7).Iron was determined calorimetrically with o-phenanthroiine, according to the directions of H. Beinert.The crystalline enzyme was dissolved in 0.1 M Na804 or in 0.2 M ammonium formate before digestion with nitric acid.Blank and standards were taken through the entire process applied to the samples.Molybdenum was determined with thiocyanate-stannous chloride (8) and copper, with dithiocarbamate (Sandell, (8) Procedure I).Assay of Enzyme-The enzyme was assayed essentially as previously described (2), except that MgClz was omitted from the reaction mixture, more buffer was used, and the preincubation of the enzyme with cysteine was prolonged.The reaction mixture for the assay contained 400 pmoles of sodium phosphate buffer of pH 6.5, 20 pmoles of freshly dissolved cysteine hydrochloride, 6 pmoles of sodium orotate, enzyme, and 0.35 pmoles