Determination of the mechanism of orotidine 5'-monophosphate decarboxylase by isotope effects.
Orotidine 5'-monophosphate shows a (15)N isotope effect of 1.0036 at N-1 for decarboxylation catalyzed by orotidine 5'-monophosphate decarboxylase. Picolinic acid shows a (15)N isotope effect of 0.9955 for decarboxylation in ethylene glycol at 190 degrees C, while N-methyl picolinic acid shows a (15)N isotope effect of 1.0053 at 120 degrees C. The transition state for enzymatic decarboxylation of orotidine 5'-monophosphate resembles the transition state for N-methyl picolinic acid in that no bond order changes take place at N-1. This rules out enolization to give a quaternary nitrogen at N-1 in the enzymatic mechanism and suggests a carbanion intermediate stabilized by simple electrostatic interaction with Lys-93. The driving force for the reaction appears to be ground-state destabilization resulting from charge repulsion between the carboxyl of the substrate and Asp-91.
Rishavy
M A
MA
The Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Cleland
W W
WW
D000641:Ammonia / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D019855:Ethylene Glycol; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen / Q000378:metabolism*; D009587:Nitrogen Isotopes; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000378:metabolism*; D010848:Picolinic Acids / Q000378:metabolism; D011522:Protons; D055672:Static Electricity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D009587:Nitrogen Isotopes; D010848:Picolinic Acids; D011522:Protons; C005417:orotidylic acid; D000641:Ammonia; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase; D019855:Ethylene Glycol; D009584:Nitrogen; C030614:picolinic acid