Analysis of the kinetic mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase.
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-line, 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 non-overlapping 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.
Hines
V
V
Department of Chemistry, Searle Chemistry Laboratory, University of Chicago, Illinois 60637.
Johnston
M
M
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004081:Dihydroorotate Oxidase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008962:Models, Theoretical; D010088:Oxidoreductases / Q000378:metabolism*
D010088:Oxidoreductases; D004081:Dihydroorotate Oxidase