Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis

Abstract

The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 10²³-fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p K_a that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p K_a = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO₂ by H⁺ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO₂ product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

Scheme 1. OMDPC-Catalyzed Decarboxylation of OMP to form UMP

Scheme 2. Partitioning of the Transition State Binding Energy for Yeast OMPDC (ScOMPDC)-Catalyzed Decarboxylation of OMP

Scheme 3. Kinetic Scheme That Describes the Activation of ScOMPDC-Catalyzed Decarboxylation of EO and FEO by Phosphite Dianion (eq 1)

Chart 1. Binding Energies ΔG^‡ for Stabilization of the Transition State for ScOMPDC-Catalyzed Decarboxylation of EO by Different Dianions, Calculated from the Dissociation Constants for Breakdown of Dianion Transition State Complexes^,

Scheme 4. Activation of ScOMPDC-Catalyzed Decarboxylation, Where ΔG^‡ is for Activator Binding to the Transition State for OMDC-Catalyzed Decarboxylation of a Truncated Substrates EO or FO (Scheme 3)

Scheme 5. Enzyme-Activating, Ligand Driven Conformational Change

Figure 1

Representations of the open (E_O) and the closed (E_C) forms of ScOMPDC. Top: Space filling models of open unliganded ScOMPDC (left, PDB entry 1DQW) and the caged complex to BMP (right, 1DQX). The Arg-235 guanidine side chain is shaded green. Middle: Representations of the open (E_O, left, 3GDK) and closed (E_C, right, 3GDL) forms of ScOMPDC. The azaUMP ligand is placed at E_O at the position determined for E_C. Bottom: Image that superimposes partial X-ray crystal structures of the OMPDC·BMP complex (PDB entry 1DQX) over the structure for unliganded ScOMPDC (1DQW). The movement of the phosphodianion gripper loop (Pro-202 to Val-220) toward the pyrimidine umbrella (Ala-151 to Thr-165) is shown. Reprinted with permission from ref (39). Copyright 2013 American Chemical Society.

Scheme 6. Effect S154A and Q215A Mutations on the Activation Barrier G^‡ (k_cat/K_m) for Wild Type ScOMPDC-Catalyzed Decarboxylation of OMP

Chart 2. Rate Constants and Changes in Activation Barriers ΔΔG^‡ for ScOMPDC-Catalyzed Decarboxylation of the Substrate Pieces (Top Row) Or the Whole Substrate OMP (Bottom Row)a

Scheme 7. Decarboxylation and Deuterium Exchange Reactions Catalyzed by ScOMPDC

Scheme 8. Phosphite Dianion Activation of the ScOMPDC-Catalyzed Deuterium Exchange Reaction of h-FEU

Scheme 9. Stabilizing Interaction between the R235 Side Chain and Transition States for ScOMPDC-Catalyzed Reactions

Scheme 10. Decarboxylation of OMP in 50:50 (v/v) H₂O/D₂O To Form h-UMP and d-UMP

Scheme 11. Mechanism for ScOMPDC-Catalyzed Decarboxylation, Which Shows Partitioning of the Vinyl Carbanion Intermediate between Direct Protonation (k_–p), and C–N Bond Rotation of the −CH₂–NL₃⁺ Group (k_rot) Followed by Transfer of a Second Hydron (k′_–p)

Scheme 12. Reversible ScOMPDC-Catalyzed Deprotonation of UMP or FUMP Followed C–N Bond Rotation of the −CH₂–NL₃⁺ Group That Leads to Exchange of the C-6 Hydrogen for Deuterium

Figure 2

Contribution of ScOMPDC gripper side chains to the intrinsic phosphodianion binding energy (eq 5) utilized for stabilization of the transition state for OMPDC-catalyzed decarboxylation.

Figure 3

Triple mutant cube that summarizes the effects (ΔΔG^‡)_OMP, in kcal/mol, of single amino acid mutations on (ΔG^‡)_OMP for the decarboxylation of OMP catalyzed by wild type ScOMPDC (black values), by single mutants of ScOMPDC (red values) and by double mutants of ScOMPDC (green values).

Scheme 13. OMPDC-Catalyzed Reactions of the Whole Substrate OMP and the Pieces EO + HP_i