BIOL 252 |
| Dihydroorotate dehydrogenases (DHOD) are flavin-containing enzymes that catalyze the oxidation of dihydroorotate (DHO) to orotate, the only redox step in the de novo synthesis of pyrimidines. During DHO oxidation, a hydride equivalent is transferred from C6 to the FMN prosthetic group and an active site base deprotonates C5 of DHO. The active site base differs between classes of DHODs – Class 1A enzymes utilize Cys while Class 2 enzymes utilize Ser – with the rest of the active site being nearly identical. A question fundamental to the mechanism of DHO oxidation is whether the scission of the two substrate C-H bonds is concerted or stepwise. This question was addressed by determining kinetic isotope effects (KIEs) on flavin reduction under anaerobic conditions. Using DHO deuterated at the 5-position, the 6-position, or both the 5- and 6-positions, KIEs were determined for the Class 2 enzyme from Escherichia coli and for the Class 1A enzyme from Lactococcus lactis. Interestingly, the two classes utilized different mechanisms of DHO oxidation. The Class 1A enzyme utilizes a concerted mechanism where the active site Cys acts as a classic general base, while the Class 2 enzyme utilizes a stepwise mechanism in which the active site Ser is a component of a proton relay system. To investigate the importance of the catalytic base in determining the mechanism of DHO oxidation, mutant enzymes in which the catalytic bases have been switched to that of the other class (S175C Class 2 enzyme from E. coli as well as C130S Class 1A DHOD from L. lactis) were studied by determining double deuterium KIEs as well as the pH dependence on flavin reduction. |
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Enzymes
4:30 PM-6:30 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster
Division of Biological Chemistry |