Mechanistic studies on the bovine liver mitochondrial dihydroorotate dehydrogenase using kinetic deuterium isotope effects

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Abstract
Dihydroorotates deuteriated at both C5 and C6 have been prepared and used to probe the mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase. Primary deuterium isotope effects on kcat are observed with both (6RS)-[5(S)-2H]- and (6RS)-[6-2H]dihydroorotates (3 and 6, respectively); these effects are maximal at low pH. At pH 6.6, DV = 3.4 for the C5-deuteriated dihydroorotate (3), and DV = 2.3 for the C6-deuteriated compound (6). The isotope effects approach unity at pH 8.8. Analysis of the pH dependence of the isotope effects on kcat reveals a shift in the rate-determining step of the enzyme mechanism as a function of pH. Dihydroorotate oxidation appears to require general base catalysis (pKB = 8.26); this step is completely rate-determining at low pH and isotopically sensitive. Reduction of the cosubstrate, coenzyme Q6, is rate-limiting at high pH and is isotopically insensitive; this step appears to require general acid catalysis (pKA = 8.42). The results of double isotope substitution studies and analysis for substrate isotope exchange with solvent point toward a concerted mechanism for oxidation of dihydroorotate. This finding serves to distinguish further the mammalian dehydrogenase from its parasitic cognate, which catalyzes a stepwise oxidation reaction [Pascal, R., and Walsh, C. T. (1984) Biochemistry 23, 2745].