Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: “Remote” Residues That Stabilize the Active Conformation

Abstract

The structural factors responsible for the extraordinary rate enhancement (~1017) of the reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and “clamps” the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a “remote” structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases kcat/Km with a minimal effect on kcat, providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5′-phosphate group of OMP for the mutant enzymes are similar to that for the wild type, supporting this conclusion.

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Importance of Residues in the Orotate Binding Site

“…However, the values of K M for the mutants are increased < 3-fold, including for the V155D mutant. Thus, each of the polar substitutions decreases the rate at which the vinyl carbanion intermediate is formed by decarboxylation but do not alter the conformational equilibrium between the open, inactive and closed, active conformations (32). …”

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

“…The four-residues engaged in the electrostatic network assume positions almost identical to those seen in the complex of WT- Mt ODCase with BMP. 17,43,44 The strong electron delocalization in effect for the O4-C4-C5-C6-O6 ‡ atoms in the flat BMP structure 17,43,44 may play a considerable role in BMP’s resistance to bond-distortion by the catalytic center.…”

“…46 More recently, Hu et al presented a mechanistic proposal that has ODCase exerting its catalytic function through direct decarboxylation to form an anionic intermediate supported by K72 stabilization of the vinyl anion intermediate of the almost decarboxylated transition state structure. 48 They also noted that the water molecule located ~2.7 Å from the O6 atom of the BMP molecule in the ODCase-BMP complex 17,43,44 must be excluded from the calculation, since no corresponding water molecules are found in all other complexes. However, all reported simulations did not consider potentially distorted substrate structures and did not analyze the interaction between the ligand and each of the protein’s residues.…”

“…ΔΔ G ‡ (difference of the transition state free energies between enzyme-catalyzed and non-enzymatic reactions) of Mt ODCase calculated from the [( k cat / K M )/ k non ] value is ca. 29–30 kcal/mol, 7,14,18,34,43,49,50 whereas that of yeast ODCase is ~31 kcal/mol. 7,51 The ΔΔ G ‡ value puts the energy required to go from the ES complex of Mt ODCase to its TS at 17–18 kcal/mol, 14,18,34,43,49,50 which is in excellent agreement with the present computational value of ~17 kcal/mol (Fig.…”

“…S1). 43,49,57–59 Various structures indicate that ligand binding leads to the closure of a loop (residues 182–190), which is disordered in the ligand-free enzyme. 6,10,19,54,55,60–62 When locked down, this loop forms five interactions, directly or through water molecules, with the phosphate part of the ligand.…”