期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 378, 期 1, 页码 84-92出版社
ACADEMIC PRESS INC
DOI: 10.1006/abbi.2000.1823
关键词
dihydroorotate dehydrogenase; substrate specificity; Saccharomyces cerevisiae; deuterium; kinetic isotope effects; flavoprotein dehydrogenase
Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity 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 (deuterated 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 deuteron of the dihydroorotate is sticky on the flavin cofactor. The pH dependencies 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, and 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 (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pK(a) of the active site base (Cys133). The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pK(a) value approximating that assigned to Cys133, Electron accepters with dihydroorotate 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 profiles versus varied concentrations of dihydroorotate with ferricyanide or O-2 as accepters suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme. (C) 2000 Academic Press.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据