OROTIDINE-5'-MONOPHOSPHATE DECARBOXYLASE CATALYSIS - KINETIC ISOTOPE EFFECTS AND THE STATE OF HYBRIDIZATION OF A BOUND TRANSITION-STATE ANALOG

The enzymatic decarboxylation of orotidine 5′-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, l -(phosphoribosyl) barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5′-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5, 6-addition of an enzyme nucleophile. When the substrate orotidine 5′-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 ± 0.06) or kcat/KM (H/D = 1.00 ± 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process. © 1990, American Chemical Society. All rights reserved.