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.

pD-Rate profile of k_ex (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 pK_a = 8.0.

We report that orotidine 5′-monophosphate decarboxylase catalyzes exchange of the C-6 proton of uridine 5′-monophosphate (UMP) for deuterium from solvent in D₂O at 25 °C (Scheme 1). Kinetic analysis of deuterium exchange gives pK_a ≤ 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.

Orotidine 5′-monophosphate decarboxylase (OMPDC) employs no metal ions or other cofactors but yet effects an enormous 1017-fold acceleration of the decarboxylation of orotidine 5′-monophosphate (OMP) to give uridine 5′-monophosphate (UMP).1 The X-ray structure of the yeast enzyme liganded with 6-hydroxyuridine 5′-monophosphate provides strong evidence that the C-6 proton of the product UMP is derived from the terminal NH₃⁺ group of Lys-93.2 The product isotope effect of unity for OMPDC-catalyzed decarboxylation of OMP in 50/50 (v/v) H₂O/D₂O eliminates a mechanism3 in which proton transfer from Lys-93 to C-6 provides electrophilic push to the loss of CO₂ in a concerted reaction.4 This result also provides strong evidence for the formation of a short-lived enzyme-bound carbanion intermediate that shows no discrimination between H and D in the proton transfer step (Scheme 2).4

The values of k_obsd (s⁻¹) determined for enzyme-catalyzed deuterium exchange in D₂O at pD 9.34 with [UMP]_total = 2.5 – 10 mM show a good fit to eq 2 that was derived for Scheme 3 (see Supporting Information), with K_d ≪ [UMP]_total. The data give the first-order rate constant for deuterium exchange into saturating enzyme-bound UMP at pD 9.34 as k_ex = 1.15 × 10⁻⁵ s⁻¹. Similar experiments using ca. 0.3 mM OMPDC (9 mg/mL) gave values of k_ex (s⁻¹) for the turnover of saturating UMP (2.5 – 5 mM) at pD 8.13 (100 mM glycylglycine buffer), and at pD 7.58 and 7.03 (48 mM imidazole buffer), at 25 °C and I = 0.1 (NaCl).11

Figure 2 shows the pD-rate profile of the values of k_ex (s⁻¹) for turnover of enzyme-bound h-UMP to give d-UMP by yeast OMPDC in D₂O at 25 °C and I = 0.1 (NaCl). The large increase in k_ex (s⁻¹) with increasing pD and the leveling off at pD > 8 shows that deuterium exchange is promoted by the basic form of an amino acid side chain at the active site of OMPDC.12 We suggest that the catalytic base is the neutral form of Lys-93,13 so that deuterium exchange arises from the reverse of the proton transfer “half reaction” that occurs in the active site during the physiological decarboxylation of OMP to give UMP (Scheme 2). Analysis of the data in Figure 2 gives p(K_a)_Lys = 8.0 for the catalytic base in D₂O at I = 0.1, and (k_ex)_max = 1.24 × 10⁻⁵ s⁻¹ for proton transfer from bound h-UMP to the neutral side chain of Lys-93 to give the enzyme-bound vinyl carbanion (Scheme 4).