Dihydroorotate Dehydrogenase from Clostridium oroticum Is a Class 1B Enzyme and Utilizes a Concerted Mechansim of Catalysis

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Abstract
Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two α (32 kDa) and two β (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from l-dihydroorotate to NAD+, defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH−kcat profile is bell-shaped with a pKa of 6.4 ± 0.1 for the ascending limb and 8.9 ± 0.1 for the descending limb; the pH−kcat/Km profile is similar but somewhat more complex. The pKa values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD+, the isotope effects on DV and D(V/KDHO), obtained upon deuteration at both the C5-proR and C5-proS positions of l-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects DV and D(V/KDHO), obtained upon additional deuteration at C6, is consistent with a mechanism in which C5-proS proton transfer and C6-hydride transfer occur in a single, partially rate-limiting step.