Analysis of the kinetic mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase

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Abstract
The steady-state kinetic mechanism of highly purified bovine liver mitochondrial dihydroorotate dehydrogenase has been investigated. Initial velocity analysis using S-dihydroorotate and coenzyme Q6 revealed parallel-like, double-reciprocal plots, indicative of a ping-pong mechanism. The dead-end inhibition pattern with barbituric acid and the reactions with alternate cosubstrates methyl-S-dihydroorotate and menadione also point to a ping-pong mechanism. However, product orotate was found to be competitive with dihydroorotate and uncompetitive with Q6. These findings suggest that dihydroorotate dehydrogenase may follow a nonclassical, two-site ping-pong mechanism, typical of an enzyme that contains two nonoverlapping and kinetically isolated substrate binding sites. That these two sites communicate by an intramolecular electron-transfer system involving FMN and perhaps an iron-sulfur center is also suggested by the kinetic behavior of the enzyme.
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