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

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