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

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

Tina L Amyes  1 , Bryant M Wood, Kui Chan, John A Gerlt, John P Richard
Affiliations

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

Tina L Amyes et al. J Am Chem Soc. .

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.

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Figures

Figure 1
Figure 1
Partial 1H NMR spectra (500 MHz) of recovered UMP obtained during exchange for deuterium of the C-6 proton catalyzed by OMPDC (0.11 mM) in D2O at pD 9.34 and 25 °C (I = 0.1, NaCl). Disappearance of the double doublet (a) due to the C-5 proton of h-UMP at 5.860 ppm (J = 8.1, 0.5 Hz, coupled to the C-6 and anomeric protons) is accompanied by the appearance of a upfield-shifted broad doublet (b) due to the C-5 proton of d-UMP at 5.857 ppm (J ≈ 0.5 Hz, coupled to the anomeric proton). The broad doublet (c) due to the anomeric proton of h-UMP at 5.889 ppm (J = 5.3 Hz, coupled to the C-2′ proton) exhibits “shoulders” (d) due to the slightly upfield-shifted doublet for the anomeric proton of d-UMP.
Figure 2
Figure 2
pD-Rate profile of kex (s−1) for turnover of enzyme-bound h-UMP to give d-UMP by OMPDC from Saccharomcyes cerevisiae (C155S mutant) in D2O at 25 °C and I = 0.1 (NaCl). The solid line shows the calculated profile for a catalytic base of pKa = 8.0.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 3
Scheme 3
Scheme 4
Scheme 4
See this image and copyright information in PMC

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