Role of the Carboxylate in Enzyme-Catalyzed Decarboxylation of Orotidine 5'-Monophosphate: Transition State Stabilization Dominates Over Ground State Destabilization

Abstract

Kinetic parameters k_ex (s^-1) and k_ex/K_d (M^-1 s^-1) are reported for exchange for deuterium in D₂O of the C-6 hydrogen of 5-fluororotidine 5'-monophosphate (FUMP) catalyzed by the Q215A, Y217F, and Q215A/Y217F variants of yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 8.1, and by the Q215A variant at pD 7.1-9.3. The pD rate profiles for wildtype ScOMPDC and the Q215A variant are identical, except for a 2.5 log unit downward displacement in the profile for the Q215A variant. The Q215A, Y217F and Q215A/Y217F substitutions cause 1.3-2.0 kcal/mol larger increases in the activation barrier for wildtype ScOMPDC-catalyzed deuterium exchange compared with decarboxylation, because of the stronger apparent side chain interaction with the transition state for the deuterium exchange reaction. The stabilization of the transition state for the OMPDC-catalyzed deuterium exchange reaction of FUMP is ca. 19 kcal/mol smaller than the transition state for decarboxylation of OMP, and ca. 8 kcal/mol smaller than for OMPDC-catalyzed deprotonation of FUMP to form the vinyl carbanion intermediate common to OMPDC-catalyzed reactions OMP/FOMP and UMP/FUMP. We propose that ScOMPDC shows similar stabilizing interactions with the common portions of decarboxylation and deprotonation transition states that lead to formation of this vinyl carbanion intermediate, and that there is a large ca. (19-8) = 11 kcal/mol stabilization of the former transition state from interactions with the nascent CO₂ of product. The effects of Q215A and Y217F substitutions on k_cat/K_m for decarboxylation of OMP are expressed mainly as an increase in K_m for the reactions catalyzed by the variant enzymes, while the effects on k_ex/K_d for deuterium exchange are expressed mainly as an increase in k_ex. This shows that the Q215 and Y217 side chains stabilize the Michaelis complex to OMP for the decarboxylation reaction, compared with the complex to FUMP for the deuterium exchange reaction. These results provide strong support for the conclusion that interactions which stabilize the transition state for ScOMPDC-catalyzed decarboxylation at a nonpolar enzyme active site dominate over interactions that destabilize the ground-state Michaelis complex.

Scheme 1. Decarboxylation Reactions Catalyzed by OMPDC

Figure 1

OMPDC-catalyzed decarboxylation of OMP and FOMP, and deuterium exchange reactions of UMP and FUMP, through common UMP or FUMP vinyl carbanion intermediates.

Figure 2

Representations of the X-ray crystal structures of ScOMPDC from Saccharomyces cerevisiae (ScOMPDC). The left-hand and middle surface structures show, respectively, the open unliganded form of ScOMPDC (PDB entry 1DQW) and the closed form with 6-hydroxyuridine 5′-monophosphate bound (PDB entry 1DQX). The phosphodianion gripper (residues 202–220) and pyrimidine umbrella loops (residues 151–165) are shaded blue, and the side chain from R235 is shaded green in both structures. The right-hand structure (PDB entry 1DQX) shows the interactions of Q215, Y217, and R235 with the phosphodianion of 6-hydroxyuridine 5′-monophosphate, and the clamping interaction between the Q215 side chain and the S154 side chain from the phosphodianion gripper and the pyrimidine umbrella loops.

Scheme 2. Kinetic Mechanism for OMPDC-Catalyzed Deuterium Exchange

Figure 3

Dependence of v_i/[E] on [FUMP] for Q215A variant ScOMPDC-catalyzed deuterium exchange reactions at I = 0.1 (NaCl). Key: (●), pD 7.1 (50 mM imidazole buffer); (▼), pD 7.4 (50 mM MOPS buffer); (⧫), pD 7.7 (50 mM MOPS buffer); (▲), pD 8.1 (50 mM GlyGly buffer); (■) pD 9.3 (50 mM GlyGly buffer).

Figure 4

Dependence of v_i/[E] on [FUMP] for ScOMPDC-catalyzed deuterium exchange reactions at pD 8.1 (50 mM GlyGly) and I = 0.1 (NaCl). (A) Y217F variant; (B) Q215A/Y217A variant.

Figure 5

Logarithmic rate profiles of kinetic parameters for ScOMPDC-catalyzed exchange for deuterium of the C-6 proton of FUMP in D₂O at 25 °C and I = 0.1 (NaCl). (A) Second-order rate constants k_ex/K_d (M^–1 s^–1) for reactions catalyzed by wild type ScOMPDC (●) and the Q215A variant (▼). (B) First order rate constants k_ex (s^–1) for reactions catalyzed by wild type ScOMPDC (●) and the Q215A variant (■). The solid lines show that fits of these data to the kinetic Scheme described previously for the reaction catalyzed by wild type OMPDC.

Figure 6

Diagram which illustrates the hypothetical interactions between ScOMPDC and late vinyl carbanion-like transition states for decarboxylation of OMP and deprotonation of FUMP. These diagrams were constructed using X-ray crystallographic data for the complex to the 6-hydroxyuridine 5′-monophosphate (BMP) transition state analog (PDB entry 1DQX), and assuming similar contacts for the stable ligand and hypothetical transition states. Not shown is stabilization of nascent CO₂ by interactions with hydrophobic amino acid side chains (see below).

Figure 7

Stepwise OMPDC-catalyzed decarboxylation of OMP (X= H, bottom reaction) and the exchange reaction of -H from FUMP for -D from solvent D₂O (X = F, top reaction). The inequality k_p ≫ k_rot for partitioning of the vinyl carbanion intermediate between proton transfer and bond rotation at the K93 side chain ensures that the deuterium enrichment of product of OMPDC-catalyzed decarboxylation of OMP is equal to the initial 50% enrichment of the mixed H₂O/D₂O solvent,^, and that the barrier to the deprotonation of FUMP (ΔG_dp^‡) is smaller than the overall barrier for the deuterium exchange reaction.

Figure 8

Hypothetical free-energy profiles for wild type and protein variants of ScOMPDC-catalyzed decarboxylation of OMP and deprotonation of UMP to form UMP vinyl carbanion intermediates UMP^-, which are drawn to show the difference in the stabilization of the respective transition states by interactions with the protein catalyst. These profiles were constructed using kinetic data from Table 1, and assuming that the changes in protein structure effect similar changes in the barriers for ScOMPDC-catalyzed deprotonation of FUMP and UMP. The diagrams show: (1) The ca. 11 kcal/mol difference in the stabilization of the transition states for wildtype ScOMPDC-catalyzed decarboxylation of OMP (ΔΔG^‡)_dc and deprotonation of UMP (ΔΔG^‡)_dp [(ΔΔG^‡)_dc – (ΔΔG^‡)_dp = 11 kcal/mol, Figure 7], that we propose is due to stabilization of the former transition state by interactions with the nascent CO₂ product. (2) The difference between the effects of Q215A or Y217F substitutions on ΔΔG_UMP^‡ (relatively large) and ΔΔG_OMP^‡ [smaller, Table 3]. (3) The larger effect of amino acid substitutions on the stability of the Michaelis complexes to OMP (ΔΔG_OMP^B) compared with UMP/FUMP (ΔΔG_FUMP^B). The ca 8 kcal/mol barrier to k_rot, which is required for the deuterium exchange reaction of UMP (k_ex) but not for decarboxylation of OMP (k_dc) or deprotonation of UMP (k_dp), is not shown. The barriers to the two enzymatic reactions have not been scaled to show the thermodynamic driving force to decarboxylation of OMP to form UMP, because this driving force is not known.

Figure 9

Representation, on the left, of the X-ray crystal structure for HsOMPDC liganded by UMP (PDB entry 2QCD) and, on the right, for ScOMPDC liganded by 6-azaUMP (PDB entry 3GDL). There is good superposition between the active site residues D312, K314, and D317 at HsOMPDC with D91, K93, and D96 at ScOMPDC, and of the hydrophobic side chains F310, I401, and I448 at the CO₂ binding pocket with F89, I183 and I232.