title: Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis.
abstract: Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
authors.forename: A
authors.initials: A
authors.lastname: Argyrou
authors.forename: M W
authors.initials: MW
authors.lastname: Washabaugh
authors.forename: C M
authors.initials: CM
authors.lastname: Pickart
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D003013:Clostridium / Q000201:enzymology*; D003903:Deuterium; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D008958:Models, Molecular; D008970:Molecular Weight; D009963:Orotic Acid / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D020836:Protein Structure, Quaternary
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D009963:Orotic Acid; D003903:Deuterium; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors