title: Purification and characterization of dihydroorotase from Pseudomonas putida.
abstract: Dihydroorotase was purified to homogeneity from Pseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-L-orotate and its methyl ester, and the reactions were reversible. The apparent Km and Vmax values for dihydro-L-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 mumol min-1 mg-1, respectively; and those for N-carbamoyl-DL-aspartate (at pH 6.0) were 2.2 mM and 68 mumol min-1 mg-1, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn2+. However, excessive Zn2+ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited by N-carbamoylamino acids such as N-carbamoylglycine, with a Ki value of 2.7 mM. The enzyme was also inhibited non-competitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with a Ki value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.
authors.affiliation: Department of Agricultural Chemistry, Kyoto University, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Ogawa
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology*; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002614:Chelating Agents; D002712:Chlorides; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D004080:Dihydroorotase; C030986:pyrimidine