Catalytic properties of dihydroorotate dehydrogenase from Saccharomyces cerevisiae: Studies on pH, alternate substrates, and inhibitors

Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogen eity from its natural source. Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same. Incubations of the yeast DHOD with dideuterodihydroorotate (deuterat ed in the positions eliminated in the dehydrogenation) as the donor and [C- 14]orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the C-14 exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 d euteron of the dihydroorotate is sticky on the flavin cofactor. The pH depe ndencies of the steady-state parameters (k(cat) and k(cat)/K-m) are similar , indicating that k(cat)/K-m reports the productive binding of substrate, a nd the parameters are dependent on the donor-acceptor pair, The lower pK(a) values for k(cat) and k(cat)/K-m observed for substrate dihydroorotate (ar ound 6) in comparison to the values determined for dihydrooxonate (around 8 ) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the an alogous hydrogen of dihydrooxonate), which is removed in the dehydrogenatio n, assists in lowering the pK(a) of the active site base (Cys133). The pH d ependencies of the kinetic isotope effects on steady-state parameters obser ved for the dideuterated dihydroorotate are consistent with the dehydrogena tion of substrate being rate limiting at low pH values, with a pK(a) value approximating that assigned to Cys133, Electron accepters with dihydroorota te as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Q(o) (0.28), fumarate (0.15), and O-2 (0.035). Orotate inhibition p rofiles versus varied concentrations of dihydroorotate with ferricyanide or O-2 as accepters suggest that both orotate and dihydroorotate have signifi cant affinities for the reduced and oxidized forms of the enzyme. (C) 2000 Academic Press.