Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata.

摘要: Deuterium-labeled dihydroorotates bearing one, two, or three deuteriums at the pair of C4 and C5 positions have been synthesized in high isotopic chiral purity characterized by NMR mass spectroscopy. These substrates used with FMN-containing biosynthetic dihydroorotate oxidase from Crithidia fasciculata [Pascal, R., Trang, N., Cerami, A., & Walsh, C. (1983) Biochemistry 22, 171] to probe stereochemistry mechanism. At pH 6.0 (4RS)-[5,5-2H2]dihydroorotate shows a Vmax isotope effect (DV) 2.83; since (4S,5R)-[5-2H]dihydroorotate DV no more than 1.1, secondary effect, overall desaturation is anti as previously reported for degradative orotate reductase Clostridium oroticum. The (4RS)-[4-2H]dihydroorotate 2.97, indicating removal C4-H also partially rate limiting 6.0. When trideuterio (4RS)-[4,5,5-2H3]dihydroorotate was tested, 8.0, value close product separate effects 4- 5S-positions, observed. this then, both C-H cleavage steps are partly catalysis. Under anaerobic conditions without an electron acceptor enzyme catalyzes preferential exchange 5S hydrogen solvent protons. aggregate on Vmax/Km [D(V/K)] analyzed suggest stepwise rather concerted mechanism pyrimidine biosynthesis.