Product Deuterium Isotope Effects for Orotidine 5'-Monophosphate Decarboxylase: Effect of Changing Substrate and Enzyme Structure on the Partitioning of the Vinyl Carbanion Reaction Intermediate

Abstract

A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-¹H]-uridine 5'-monophosphate (50%) and [6-²H]-uridine 5'-monophosphate (50%) from the decarboxylation of orotidine 5'-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) from S. cerevisiae, M. thermautotrophicus and E. coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO₂ in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, that is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH₂-NL₃⁺ group of the side-chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5'-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. The following mutations of important active site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A and S154A/Q215A.

Introduction

Orotidine 5'-monophosphate decarboxylase (OMPDC) is a remarkable enzyme because it employs no metal ions or other cofactors, but yet it effects an enormous ca. 30 kcal/mol stabilization of the transition state for the decarboxylation of orotidine 5'-monophosphate (OMP) to give uridine 5'-monophosphate (UMP, Scheme 1).^1–3 A significant fraction of this rate acceleration is obtained from utilization of the intrinsic phosphate binding energy in stabilization of the decarboxylation transition state.^4,5 The electrophilic substitution of -CO₂ by a proton may proceed by two reaction steps or stages: loss of CO₂ to form the putative vinyl carbanion reaction intermediate and protonation of this carbanion by an acidic catalytic residue to form UMP. It has also been suggested that these two steps might be coupled in a concerted electrophilic substitution reaction.^6–8

Scheme 1.

OMPDC is assayed in the direction of decarboxylation of OMP, and mechanistic studies have naturally focused on the loss of CO₂ from substrate, which is the first stage in a stepwise reaction mechanism. The second stage of proton transfer between the enzyme and substrate has received much less consideration in experimental and theoretical studies.^2,9 This strong focus on the first reaction stage has resulted in a significant, but unappreciated, gap in our understanding of the enzymatic reaction mechanism, which can only be fully defined by studies that encompass the whole reaction.

Loss of CO₂ and the addition of a proton to carbon-6 of OMP may proceed by the fully stepwise reaction mechanism shown in Scheme 1, or by a concerted mechanism that avoids the formation of the vinyl carbanion reaction intermediate. The latter was suggested for OMPDC-catalyzed decarboxylation, in order to provide a mechanism for avoiding formation of the unstable vinyl carbanion intermediate, while at the same time providing for transition state stabilization from electrophilic push by the transferred proton.^6–8 This distinction between stepwise and concerted reaction mechanisms for proton transfer from enzyme to substrate can only be made by a direct study of the OMPDC-catalyzed proton transfer reaction.

The product deuterium isotope effect (PIE, Scheme 2) determined directly as the yield of products of reaction of -H and of -D in 50/50 HOH/DOD provides a measure of the relative electrophilic push of the transferred –H and –D at the transition state for chemical and enzymatic reactions. We have determined PIEs of as large as 8 for thermoneutral proton transfer from carboxylic acids to ring-substituted α– methoxystyrenes, where there is apparently the full loss of zero-point energy at the transferred hydron at the rate-determining transition state.^10,11 The PIE decreases below this maximum value for either thermodynamically favorable or unfavorable proton transfer reactions.^11–13 PIEs of 1.0 for nonenzymatic reactions in aqueous solution are almost never observed,^14–18 but are possible for a complex reaction mechanism where proton transfer occurs after the step that determines the yields of hydrogen and deuterium labeled reaction products. In this case the deuterium enrichment of product will be determined by the initial deuterium enrichment of the catalytic acid.

Scheme 2.

We recently reported a fast and convenient method to determine the yields of [6-¹H]-uridine 5'-monophosphate ([6-¹H]-UMP]) and [6-²H]-uridine 5'-monophosphate ([6-²H]-UMP]) from the decarboxylation of OMP in 50/50 (v/v) HOH/DOD catalyzed by OMPDC. The ratio of the yields of these two products was determined to be equal to the isotopic enrichment of solvent, so that the PIE on this reaction is 1.0.¹⁹ We now report: (a) the full experimental details from our earlier preliminary communication and a significant modification of the protocol for ¹H NMR analysis of the deuterium enrichment of product UMP; (b) the PIE on OMPDC-catalyzed decarboxylation of 5-FOMP a substrate that is activated for decarboxylation by the C-5 fluorine; and, (c) PIEs on decarboxylation of OMP and 5-FOMP by several site-directed mutants of OMPDC. These results provide strong evidence that the step that determines the yields of H- and D-labeled products is decarboxylation of enzyme-bound substrate, and that protonation of the carbanion intermediate of decarboxylation is faster than steps that would exchange acidic hydrons and allow the intermediate to select between reaction with –H and –D. The observed PIE shows a remarkable insensitivity to changes in substrate reactivity and enzyme catalytic activity.

Experimental

Materials

Orotidine 5'-monophosphate trisodium salt (99%) was purchased from Sigma or was prepared by chemical methods from uridine 5'-monophosphate using modifications of literature procedures.^20–22 3-(N-morpholino)propanesulfonic acid (MOPS, ≥ 99.5%) was purchased from Fluka. Water was from a Milli-Q purification system. Deuterium labeled water (99.9% D), DCl (35% wt, 99.9%D) and KOD (40% wt, > 98%D) were from Cambridge Isotope Laboratories. 5-FOMP was prepared by orotate phosphoribosyl transferase-catalyzed coupling of 5-fluoroorotate and 5’-phosphoribosyl-1’-pyrophosphate by following a literature procedure.²³ All other chemicals were reagent grade or better and were used without further purification.

The S155C mutant of OMPDC from Saccharomyces cerevisiae (ScOMPDC) was prepared as described in earlier work.^23,24 This enzyme was used as a model for the wildtype enzyme, because the mutant is more stable than, but kinetically and structurally essentially identical with wildtype ScOMPDC. OMPDC from Methanothermobacter thermautotrophicus (MtOMPDC) and from Escherichia coli (EcOMPDC) were also prepared and purified as described in earlier work.^23,25 The gene for S155C mutant of ScOMPDC was used as the starting point for preparation of the S154A,²⁴ Q215A,²⁴ S154A/Q215A,²⁴ and R235A²⁶ mutant enzymes. Similar procedures were followed in preparing the Y217A mutant enzyme of ScOMPDC [see Supporting Information].

NMR Analyses

¹H NMR spectra (ca 30 transients) were recorded on a Varian Unity Inova-500 spectrometer using a sweep width of 6000 Hz, a 90° pulse angle, an acquisition time of 6 s, a relaxation delay between pulses of 120 s (> 7T₁) and with suppression of the water peak. Baselines were subjected to first-order drift corrections before integration of the signals. Chemical shifts are reported relative to HOD at 4.67 ppm. ¹⁹F NMR spectra (64 transients) were recorded on a Varian Unity Inova-500 spectrometer using a spectral width of 50000 Hz, a 90° pulse angle, a 2.6 s acquisition time and a relaxation delay between pulses of 20 s (> 7T₁). The receiver gating time was optimized to obtain a flat baseline. Baselines were subject to first-order drift correction before integration of the signals. Chemical shifts are reported relative to a neat trifluoracetic acid external standard for which δ ™= −78.5 ppm.²⁷

OMPDC-Catalyzed Decarboxylation

Stock solutions of OMP and 5-FOMP were prepared in 50/50 HOH/DOD and their concentrations were determined from the absorbance in 0.1 M HCl at 267 nm using ε = 9400 M⁻¹ cm⁻¹ and 10200 M⁻¹ cm⁻¹ for OMP and 5-FOMP, respectively. The concentration of wildtype ScOMPDC, EcOMPDC and MtOMPDC in stock solutions was determined from the absorbance at 280 nm using values of ε = 29900, 10100 and 6100 M⁻¹ cm⁻¹,²⁵ respectively calculated using the ProtParam tool available on the ExPASy server.^28,29 The concentration of mutant OMPDCs in stock solutions was determined from the absorbance at 280 nm using ε = 29900 M⁻¹ cm⁻¹ or ε = 28420 M⁻¹cm⁻¹ for the Y217A mutant calculated using the ProtParam tool. All enzyme assays were carried out at 25 °C in 10 mM MOPS (50% free base) at pH 7.1 and I = 0.105 (NaCl). Standard assays described in earlier work were used to determine the activity of OMPDC for decarboxylation of OMP.²⁴

Decarboxylation of OMP (2 mM) catalyzed by wildtype and mutant OMPDCs at 25 °C was carried out in 50/50 HOH/DOD (2 mL) that contained 50 mM MOPS buffer (50% free base) at pL 7.4 with ionic strength of 0.1 (NaCl). The reactions were initiated by addition of OMPDC (0.01 – 30 µM). The reaction was allowed to proceed until ca 95% complete, at which time (x min) an 800 µL aliquot was withdrawn and the enzyme was removed by ultrafiltration using Microcon® Centrifugal Filter Units (10K MWCO). After an additional reaction time of x min a second 800 µL aliquot was withdrawn and treated in the same manner. The filtered samples were frozen, until NMR analysis of the deuterium enrichment of the 6-H position. Prior to ¹H NMR analyses, the solution pL was adjusted to 9.5 – 10 using NaOD, in order to increase the resolution of the signals for the 5-H and 1’-H of UMP. The same procedures were followed in studies of OMPDC-catalyzed decarboxylation of 5-FOMP (2 mM), except that the pL was kept at the initial value of 7.4 for ¹⁹F NMR analyses.

Results

The yields of H- and D-labeled products from decarboxylation of OMP catalyzed by OMPDC in 50/50 (v/v) HOH/DOD (Scheme 3, X = H) were determined by ¹H NMR spectroscopy at 500 MHz. Figure 1A shows a representative partial ¹H NMR spectrum at pL 7.4 of UMP obtained from the decarboxylation of OMP (2 mM) catalyzed by ScOMPDC (C155S mutant, 0.024 µM, 200 min reaction time) in 50/50 (v/v) HOH/DOD at pL 7.4 and 25 °C (I =0.10, NaCl). ¹⁹ The product deuterium isotope effect of 1.0 (Table 1) was determined as the ratio of the integrated area A_H of the doublet due to the C-6 proton of [6-¹H]-UMP at 7.990 ppm and A_D of the collapsed singlet due to the C-5 proton of [6-²H]-UMP at 5.865 ppm (eq 1).¹⁹

$$\text{PIE}=\frac{[{\mathrm{P}}_{\mathrm{H}}]}{[{\mathrm{P}}_{\mathrm{D}}]}=\frac{A_H}{A_D}$$ (1)

Scheme 3.

Figure 1.

(A) Partial ¹H NMR spectrum (500 MHz) at pL 7.4 of UMP from decarboxylation of OMP (2 mM) catalyzed by ScOMPDC (0.024 µM) in 50/50 (v/v) HOH/DOD at 25 °C. Key: (■), Doublet due to the C-6 proton of [6-¹H]-UMP; (●), double doublets (poorly resolved) due to the anomeric protons of [6-¹H]-UMP and [6-²H]-UMP; (♦), doublet due to the C-5 proton of [6-¹H]-UMP; (▼), collapsed singlet due to the C-5 proton of [6-²H]-UMP. (B) Partial ¹H NMR spectrum at pL 9.7 of [6-¹H]-UMP and [6-²H]-UMP. Key: (●), Double doublets due to the anomeric protons of [6-¹H]-UMP and [6-²H]-UMP; (♦), doublet due to the C-5 proton of [6-¹H]-UMP; (▼), collapsed singlet due to the C-5 proton of [6-²H]-UMP.

Table 1.

Product Deuterium Isotope Effects for Catalysis of Decarboxylation of OMP and 5-FOMP by Wildtype and Mutants of Orotidine 5-Monophosphate Decarboxylase.^a

Enzyme		kcat/Km (M−1 s−1)	Substrate	[E]/µM	Reaction Time (min) b	PIE c
E. coli	Wildtype	6.4 × 105	OMP	0.044	200, 400	1.05 ± 0.04 d
M thermautotrophicus	Wildtype	3.3 × 106	OMP	0.042	600, 1200	1.01 ± 0.07 d
S. cerevisiae	Wildtype	1.1 ×107	OMP	0.024	200	1.0 d
				0.47	17, 33	1.02 ± 0.01 e
		1.2 × 107	5-FOMP	0.13	8, 16	0.93 ± 0.02 f
	R235A	610	OMP	13	15, 30	1.03 ± 0.01 e
		1.3 × 105	5-FOMP	0.054	15, 30	0.92 ± 0.04 f
	Q215A	2.5 × 105	OMP	0.026	200, 400	1.06 ± 0.01 e
		2.6 × 106	5-FOMP	0.010	40, 80	0.95 ± 0.01 f
	S154A	630	OMP	28	55, 110	1.02 ± 0.02 e
		2.7 × 105	5-FOMP	1.6	5, 10	0.95 ± 0.02 f
	S154A/Q215A	380	OMP	12	240, 480	1.04 ± 0.01 e
		7.7 × 104	5-FOMP	0.38	150, 300	0.92 ± 0.01 f
	Y217A	2500	OMP	0.59	50, 100	1.03 ± 0.02 e
		1.3 × 104	5-FOMP	0.40	45, 90	0.93 ± 0.02 f

^aFor reactions in 50/50 (v/v) HOH/DOD at pL 7.4 and 25 °C (I = 0.10, NaCl).

^bThe product yields were first determined after ca 95% completion of the decarboxylation reaction (see text).

^cThe ratio of the yields of the -H and -D labeled products for reactions in 50/50 (v/v) HOH/DOD. In all cases but one, the reported PIEs are the average of the values determined at two different reaction times. The quoted uncertainty gives the range of these two values.

^dThe ratio of the area from ¹H-NMR of the doublet for the C-6 hydrogen of [6- ¹H]-UMP and of the collapsed singlet for the C-5 hydrogen of [6-²H]-UMP determined from spectral data at pL 7.4 (Figure 1A).

^eThe ratio of the area from ¹H-NMR of the doublet for the C-5 hydrogen of [6-¹H]-UMP and of the collapsed singlet for the C-5 hydrogen of [6-²H]-UMP determined from spectral data at pL 9.5 – 10 (Figure 1B).

^fThe ratio of the area from ¹⁹F-NMR of the doublet for the C-5 fluorine of [6-¹H]-UMP and of the singlet for the C-5 fluorine of [6-²H]-UMP (Figure 2).

The spectrum of UMP at pL 9.7 shows a better resolution of the signals for the C-5 pyrimidine and the C-1’ anomeric hydrogen, because of the effect of partial ionization of N-3 of UMP (pK_a = 9.45)³⁰ on chemical shift. Figure 1B shows a representative partial ¹H NMR spectrum, at pL 9.7, of the UMP product of decarboxylation of OMP (2 mM) catalyzed by the R235A mutant of ScOMPDC (13 µM) in 50/50 (v/v) HOH/DOD at pL 7.4 and 25 °C (I = 0.10, NaCl) and a reaction time of 30 min. The PIE of A_H/A_D = 1.03 ± 0.01 was calculated from the ratio of the integrated area of the doublet at 5.846 ppm for the C-5 hydrogen of [6-¹H]-UMP and of the collapsed singlet at 5.843 ppm for the C-5 hydrogen of [6-²H]-UMP. This PIE is the average of the values determined at two different reaction times, and the quoted uncertainty gives the range of the two experimental values.

Table 1 reports product deuterium isotope effects on the decarboxylation of OMP (2 mM) at pL 7.4 catalyzed by wildtype OMPDC from several different organisms and by several mutant forms of OMPDC from S. cerevisiae. These PIEs were calculated as the ratio of the integrated area of the C-5 hydrogen of [6-¹H]-UMP and of the C-5 hydrogen of [6-²H]-UMP determined for spectra at pL 9.5 – 10, similar to the spectrum shown in Figure 1B. There is good agreement (± 5%) between the PIEs calculated from spectral data at pL 7.4 (Figure 1A) and at pL 9.5 – 10. We have used data from NMR spectra determined at the higher pL, because of the better resolution of peaks for the relevant protons. Table 1 reports PIEs that are the average of values determined at two reaction times. The first time (x min) was after ca 95% completion of the decarboxylation reaction. A second analysis at 2x min was carried out to verify that the reaction product UMP was stable under the reaction conditions to further OMPDC-catalyzed hydron exchange,^31,32 and to provide an estimate for the uncertainty in these product isotope effects. In every case but one, the agreement between the PIEs determined from the products obtained at these two reaction times is better than ± 5%.

Figure 2 shows a representative ¹⁹F-NMR spectrum (500 MHz) at pL 7.4 of 5-FUMP obtained from the decarboxylation of 5-FOMP (2 mM) catalyzed by R235A mutant ScOMPDC (0.054 µM in 50/50 (v/v) HOD/DOD) at pL 7.4 and 25 °C (I = 0.10, NaCl) and a reaction time of 30 min. Note that the deuterium at C-6 of [6-²H]-5-FUMP perturbs the ¹⁹F-chemical shift of the C-5 fluorine by 0.29 ppm. The product deuterium isotope effect of 0.92 was determined as the ratio of the integrated area of the doublet at δ= 47.70 ppm (J_HF = 6 Hz) due to the fluorine at [6-¹H]-5-F-UMP and of the downfield-shifted singlet at δ = 47.41 due to the C-5 fluorine at [6-²H]-5-FUMP (Scheme 2, X = F).

Figure 2.

¹⁹F NMR spectrum of 5-FUMP, which is the product of decarboxylation of 5-FOMP catalyzed by R235A ScOMPDC (0.054 µM) at pL 7.4. The reported chemical shifts are relative to a neat trifluoracetic acid external standard for which δ= −78.5 ppm.²⁷ Key: (Δ), doublet at δ=47.70 ppm (J_HF = 6 Hz) due to the fluorine at [6-¹H]-5-FUMP; (♦), downfield-shifted singlet at δ=47.41 ppm due to the fluorine at [6-²H]-5-FUMP.

Table 1 reports the PIEs on decarboxylation of 5-FOMP (2 mM) at pL 7.4 catalyzed by wildtype ScOMPDC and by several mutant forms of ScOMPDC. These PIEs were calculated as the ratio A_H/A_D of the integrated area of C-5 fluorine of [6-¹H]-FUMP and of the C-5 fluorine of [6-²H]-FUMP determined for spectra similar to the spectrum shown in Figure 2. The PIEs reported in Table 2 are the average of values determined at two reaction times. The first time (x min) was after ca 95% completion of the decarboxylation reaction. A second analysis at 2x min was carried out to verify that the product 5-FUMP was stable under the reaction conditions to further OMPDC-catalyzed hydron exchange.^31,32 The agreement between the PIEs determined for the products obtained at these two reaction times is better than ±5%.

Discussion

The product deuterium isotope effect on electrophilic substitution reactions of the proton in aqueous solution depends strongly on the lifetime of the putative carbanion intermediate of the stepwise reaction.³³ A PIE of 2.8 was determined for addition of the mercaptoethyl thiolate anion to acrylonitrile in water (Chart 1A).¹⁸ This reaction proceeds through the α–cyano stabilized carbanion 1 that shows a modest selectivity for protonation by HOL compared with DOL.³⁴ The sharp decease from 10 to 1.2 in the PIE on methoxide ion-catalyzed cleavage of substituted benzyltrimethylsilanes in 50/50 MeOH/MeOD (Chart 2B) is due to the large decrease in the selectivity of the strongly resonance stabilized carbanion 4-NO₂-2compared to the much more unstable unsubsituted benzylic carbanion H-2.¹⁷ Finally, the PIE of 1.9 determined for specific base-catalyzed retroaldol cleavage of 1-phenylcyclopropanol reflects the stronger electrophilic push of HOL compared with DOL for C-C bond cleavage by a concerted reaction mechanism. This concerted mechanism is enforced because the putative primary carbanion intermediate 3 of the stepwise mechanism is far too unstable to exist for the time of a bond vibration in water.^33,35

Chart 1.

X-ray crystallographic analysis of the complex between ScOMPDC and the transition state analog 6-hydroxyuridine 5'-monophosphate (BMP) shows that the acidic ε-NH₃ ⁺ group of the Lys-93 appears to be positioned to protonate the putative C-6 carbanion reaction intermediate of decarboxylation of OMP.³¹ The PIE on OMPDC-catalyzed decarboxylation is equal to 1/Ф_TS (eq 2, Scheme 4), where Ф_TS is the fractionation factor for partitioning of –H and –D between solvent LOL and the ε-NL₃ ⁺ group of the Lys-93 at the transition state for decarboxylation.^36–38 The kinetic deuterium isotope effect (KIE = k_EH/k_ED, Scheme 4) on OMPDC-catalyzed decarboxylation of OMP in 50/50 HOH/DOD is controlled by the fractionation factor Ф_EL for partitioning of -H and -D between solvent L-OL and the ε-NL₃ ⁺ group of the Lys-93, and by Ф_TS(eq 3, Scheme 4). Values of Ф_EL ≈ 1.0 have been reported for H/D fractionation between L₂O and R-NL₃ ⁺,^36,39 so that the product isotope effect (PIE) of 1.0 is equal to the kinetic isotope effect (KIE).

$$(\text{PIE})=1/{\mathrm{\Phi }}_{\text{TS}}$$ (2)

$$(\text{KIE})=\left(\frac{k_{\text{EH}}}{k_{\text{ED}}}\right)=\left(\frac{K_{\text{EH}}^{\pm }}{K_{\text{ED}}^{\pm }}\right)=\frac{{\mathrm{\Phi }}_{\text{EL}}}{{\mathrm{\Phi }}_{\text{TS}}}={\mathrm{\Phi }}_{\text{EL}}(\text{PIE})$$ (3)

Scheme 4.

The values of PIE = KIE = 1.0 for decarboxylation of OMP catalyzed by wildtype ScOMPDC, EcOMPDC and MtOMPDC show that there is no selectivity between the reaction of –H and –D at the transition state for the rate-determining step. Therefore, the zero-point energy of the bond to the transferred hydron does not change significantly on proceeding first from the ground state of 50/50 HOH/DOD to the ε-NL₃ ⁺ of Lys-93, and then to the transition state for the rate-determining step for the OMPDC-catalyzed decarboxylation reaction. The absence of any significant primary deuterium isotope effect shows that the rate-limiting step for OMPDC catalyzed decarboxylation is strongly uncoupled from the proton transfer step.

It is interesting that essentially the same PIE is observed for OMPDCs from S. cerevisiae, M. thermautotrophicus and E. coli (Table 1). These three OMPDCs show striking differences in the size and structure of a flexible loop that interacts with the phosphodianion of bound substrate and provides binding energy that is utilized in catalysis of decarboxylation of OMP,^4,25 but they otherwise exhibit a high degree of structural homology.⁶ The results reported here show that the PIEs on these OMPDC-catalyzed reactions are not controlled by the size of the enzyme flexible phosphate gripper loops.

The PIE of 1.0 for decarboxylation of OMP is inconsistent with a transition state for a concerted mechanism in which there is significant electrophilic “push” by the transferred hydron to displacement of CO₂.^6–8 The observation that OMPDC catalyzes the slow exchange of the C-6 hydrogen of UMP for deuterium from solvent³¹ and of the ca 1000-fold faster exchange of the C-6 hydrogen of 5-FUMP ³² show that OMPDC provides strong stabilization of a bound UMP carbanion. They provide strong support for formation of the UMP carbanion as an intermediate of the decarboxylation reaction.³¹ The PIEs reported in this work cannot be easily rationalized by a stepwise reaction mechanism in which the lifetime of a UMP carbanion reaction intermediate is sufficiently long to allow for it to “select” between reaction with –H or –D from the enzyme. This is because there is a significant discrimination between protonation of carbanions by –H and –D, even when proton transfer is strongly thermodynamically favorable.¹⁸

We propose that breakdown of OMP to form a C-6 UMP carbanion intermediate is the rate-determining step for OMPDC-catalyzed decarboxylation, and that hydron transfer from the side-chain of Lys-93 to this vinyl carbanion is faster than movement which exchanges the positions of the N-L⁺ hydrons and allows the carbanion to select for reaction with H or D (k_−p >> k_r, Scheme 5). The yield of [6-¹H]-UMP and [6-²H]-UMP products is therefore determined by the initial enrichment of this acid in –H and –D (Ф_EL, Scheme 4).

Scheme 5.

These results are consistent with the observed -¹³CO₂ ⁻ kinetic isotope effect of 1.0255 on k_cat/K_m for OMPDC-catalyzed decarboxylation of OMP,²¹ which shows that C-C bond cleavage partly limits the overall rate of this decarboxylation reaction.^21,40 The decrease to 1.0106 in the [¹³C]-KIE on k_cat/K_m for OMPDC-catalyzed decarboxylation of [6-¹³C]-5-FOMP is consistent with a partial change in rate determining step, from C-C bond cleavage to substrate binding, for decarboxylation of this activated substrate.²¹ The value of the product isotope effect is determined by the rate constants for partitioning of the carbanion intermediate between reaction with –H and with –D, and it is not affected by this change in rate determining step for the overall decarboxylation reaction.

The rate constant for C-NL₃ ⁺ bond rotation in water is similar to the value of 10¹¹ s⁻¹ estimated for reorganization that exchanges the relative positions of –O and –S at a thiobenzoate•carbocation ion pair in water. ⁴¹ The X-ray crystal structure of yeast OMPDC complexed with BMP shows that the -CH₂-NH₃ ⁺ group of Lys-93 is fixed by hydrogen bonds to the carboxylate groups of Asp-91 and Asp-96 (Scheme 5).⁴² We propose that these two hydrogen bonds restrict rotation about the carbon-nitrogen bond of the CH₂-NL₃ ⁺ group of Lys-93, so that k_r << 10¹¹ s⁻¹; and, that they direct the single unliganded hydron towards the C-6 UMP carbanion. Decarboxylation of bound OMP (Scheme 5) then results in a deuterium enrichment of product UMP that is essentially the same as the enrichment of CH₂-NL₃ ⁺ in 50/50 HOH/DOD, because of the strong forward commitment for transfer of the non-hydrogen bonded hydron of Lys-93 to the C-6 UMP carbanion (k_−p >> k_r, Scheme 5).

Effect of Changing Reactivity of Substrate and Enzyme

The PIE of 0.93 for OMPDC-catalyzed decarboxylation of 5-FOMP by wildtype ScOMPDC is ca. 10% smaller than the value of 1.02 determined for wildtype OMPDC-catalyzed decarboxylation of OMP (Table 1). Similar 5–10% differences in PIEs for OMPDC-catalyzed decarboxylation of 5-FOMP and OMP are also observed for the reactions catalyzed by several mutant enzymes. These differences are reproducible and larger than the uncertainty in the individual PIEs.

The addition of the 5-F to OMP causes only small changes in k_cat/K_m (from 1.3×10⁷ to 2.6×10⁷ M⁻¹ s⁻¹) and in k_cat (from 20 to 130 s⁻¹) for ScOMPDC-catalyzed decarboxylation.²³ However, the full effect of the 5-fluorine substituent on the microscopic rate constant for decarboxylation of bound substrate is not observed, because binding of 5-FOMP to OMPDC is strongly rate determining for k_cat/K_m and release of product 5-FUMP is strongly rate determining for k_cat.²³ By comparison, addition of the 5-fluorine to OMP causes a much larger 200-fold increase in k_cat/K_m (from 610 to 1.2×10⁵ M⁻¹ s⁻¹, Table 1) and 270-fold increase in k_cat (from 1.2 to 320 s⁻¹)⁴³ for decarboxylation of OMP catalyzed by the R235A mutant of ScOMPDC. This shows that the 5-fluorine provides ca 3 kcal/mol stabilization of the transition state for OMPDC-catalyzed decarboxylation when the breakdown of enzyme-bound 5-FOMP is the rate-determining step. This substituent provides a similar stabilization of the UMP carbanion intermediate of the OMPDC-catalyzed deuterium exchange reaction of UMP.³²

Stabilization of the OMPDC-bound UMP carbanion by the 5-fluorine substituent should lead to a decrease in k_−p (Scheme 5) for protonation of this intermediate.⁴⁴ The PIE of 0.93 for decarboxylation of 5-FOMP catalyzed by wildtype ScOMPDC (Table 1) shows, not only that this decrease in k_−p is not sufficiently large to affect the inequality k_−p >>k_r (Scheme 5) that was proposed for partitioning of the UMP vinyl carbanion, but that the PIE has become smaller than the limiting KIE of 1.0 for a reaction where there the is no movement of hydrogen on moving from reactant to transitions state. We propose that the PIE of 0.93 is observed because the deuterium enrichment of the product 5-FUMP is the same as the enrichment of CH₂-NL₃ ⁺ at the OMPDC•5-FOMP Michaelis complex. This enrichment is measured by the fractionation factor Ф_EL (PIE = KIE/Ф_EL, Scheme 4). The smaller PIE on wildtype ScOMPDC-catalyzed decarboxylation of 5-FOMP (0.93 ± 0.02) compared to decarboxylation of OMP (1.02 ± 0.02, Table 1) is consistent with an increase in Ф_EL from 1.0 for decarboxylation of OMP to 1.1 for decarboxylation of 5-FOMP. This increase in Ф_EL provides evidence that effect of the 5-fluorine at enzyme-bound 5-FOMP is to strengthen the N-L bonds of the -CH₂-NL₃ ⁺ group of Lys-93.^37,38 This may be due to an interaction between the negative end of the C-F dipole and positively charged -CH₂-NL₃ ⁺ that stabilizes the alkylammonium ion relative to the basic amine.

The PIEs on decarboxylation of OMP and 5-FOMP catalyzed by OMPDC show a remarkable insensitivity to mutation of amino acid residues that are essential for the observation of robust enzymatic activity (Table 1). In other words, these mutations affect the stability of the rate-determining transition state for decarboxylation, but do not affect the relative yield of –H and –D labeled reaction products. The results are consistent with the mechanism shown in Scheme 5, where k_−p >> k_r for all mutant enzyme-catalyzed reactions. This is simply rationalized. The active site residues examined all lie distant from Lys-93 and so their mutation is not expected to affect the barrier to k_r for rotation about the carbon-nitrogen bond of the -CH₂-NL₃ ⁺ group of Lys-93. The mutations all cause an increase in the barrier for decarboxylation of OMP to form the C-6 UMP carbanion. This presumably reflects an increase in the barrier to formation of the carbanion intermediate, which should be accompanied by a decrease in the activation barrier to carbanion protonation (increase in k_−p, Scheme 5)⁴⁴ that will favor observation of a unitary PIE.

There is a striking contrast between the large conformational flexibility of OMPDC that is suggested by the differences in the structures for the free and liganded enzyme,⁴² and the rotational rigidity of -CH₂-NL₃ ⁺ group of Lys-93 that is hydrogen-bonded to carboxylate groups of Asp-91 and Asp-96 (Scheme 5).⁴² We note that the most efficient catalysis of decarboxylation will be observed when there is the maximum increase in stabilizing electrostatic interactions between the -CH₂-NL₃ ⁺ group of OMPDC and the bound substrate/intermediate on proceeding from the Michaelis complex to the C-6 UMP carbanion reaction intermediate. Therefore, one role for “clamping” the -CH₂-NL₃ ⁺ side-chain of Lys 93 may be to position this amino acid side chain to minimize its electrostatic interactions with the -CO₂ ⁻ of OMP in the Michaelis complex and to maximize the interactions with the developing C-6 UMP carbanion at the transition state for decarboxylation.^45,46 This may involve a shift in the position of the side-chain that is coupled in some way to decarboxylation.