Formation and stability of a vinyl carbanion at the active site of orotidine 5'-monophosphate decarboxylase: pKa of the C-6 proton of enzyme-bound UMP

J Am Chem Soc. 2008 Feb 6;130(5):1574-5. doi: 10.1021/ja710384t. Epub 2008 Jan 11.

Abstract

We report that orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes exchange of the C-6 proton of uridine 5′-monophosphate (UMP) for deuterium from solvent in D2O at 25 °C and pD 7.0 – 9.3. Kinetic analysis of deuterium exchange gives pKa ≤ 22 for carbon deprotonation of enzyme-bound UMP, which is at least 10 units lower than that for deprotonation of an analog of UMP in water. The observation of enzyme-catalyzed deuterium exchange via a stabilized carbanion provides convincing evidence for the decarboxylation of orotidine 5′-monophosphate (OMP) by OMPDC to give the same carbanion intermediate. The data show that yeast OMPDC stabilizes the bound vinyl carbanion by at least 14 kcal/mol. We conclude that OMPDC also provides substantial stabilization of the late carbanion-like transition state for the decarboxylation of OMP, and that this transition state stabilization constitutes a large fraction, but probably not all, of the enormous 10-fold enzymatic rate acceleration.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Carbon / chemistry*
  • Magnetic Resonance Spectroscopy
  • Molecular Structure
  • Orotidine-5'-Phosphate Decarboxylase / metabolism*
  • Protein Binding
  • Protons*
  • Uridine Monophosphate / chemistry*
  • Uridine Monophosphate / metabolism*
  • Vinyl Compounds / chemistry*

Substances

  • Protons
  • Vinyl Compounds
  • Carbon
  • Uridine Monophosphate
  • Orotidine-5'-Phosphate Decarboxylase