Orotidine-5'-monophosphate decarboxylase catalysis: kinetic isotope effects and the state of hybridization of a bound transition-state analogue.
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, 1-(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.
Acheson
S A
SA
Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27599.
Bell
J B
JB
Jones
M E
ME
Wolfenden
R
R
D002262:Carboxy-Lyases / Q000378:metabolism*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003903:Deuterium; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
D003903:Deuterium; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase