BIOL 245 |
| Phosphoenolpyruvate carboxykinases catalyze the carboxylation-dephosphorylation of phosphoenolpyruvate in the presence of Mn2+, CO2, and a nucleoside diphosphate to yield oxaloacetate and the nucleoside triphosphate. The stereochemical course of the carboxylation reaction catalyzed by ATP-dependent enzymes has not been determined, although it is known that CO2 addition occurs by the si face of phosphoenolpyruvate when catalyzed by GTP-dependent phosphoenolpyruvate carboxykinases [Huang and Nowak (1986) Biochemistry 25, 5590-5595]. In this work we show that the ATP-dependent phosphoenolpyruvate carboxykinases from Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens can use (Z)-3-fluorophosphoenolpyruvate as a substrate analogue. The produced fluorooxaloacetate was reduced to fluoromalate, and 19F NMR analyses showed that it corresponds to (2R,3R)-3-fluoromalate, thus indicating that the addition of CO2 to (Z)-3-fluorophosphoenolpyruvate takes place by the si face. These results show that ATP-dependent and GTP-dependent phosphoenolpyruvate carboxykinases, in spite of their very different primary structure, catalyze the carboxylation reaction with the same stereochemistry. Supported by FONDECYT 1070202-1030760. |
|
Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |