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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem, Biophys. 146, 256-270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000. In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor. Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo. Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine methosulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. & Kennedy, J. A.: J. Biol. Chem. 250, 4322-4326 (1975)).
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PMID:A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme. 13 Jan 99

Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].
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PMID:Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei. 629 33