Electrostatic stress in catalysis: Structure and mechanism of the enzyme orotidine monophosphate decarboxylase

Abstract

Orotidine 5 -monophosphate decarboxylase catalyzes the conversion of orotidine 5 -monophosphate to uridine 5 -monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5 -monophosphate-complexed forms have been determined at 1.8 and 1.5 Å, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5 -monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.O rotidine 5Ј-monophosphate decarboxylase (ODCase) (EC 4.1.1.23) formally catalyzes the exchange of CO 2 for a proton at the C 6 position to form uridine 5Ј-monophosphate (UMP) (1). The intermediate implied by this description consists of a C 6 -carbanion, the conjugate base of the UMP carbon acid. The ODCase reaction is unique in biological decarboxylation reactions in that the carbanion intermediate is not stabilized by conjugation interactions and, thus, the reaction rate is exceptionally slow in aqueous solution (2). The remarkable catalytic power of ODCase, which accelerates the reaction by 17 orders of magnitude over the aqueous process, has fascinated chemists and biochemists alike, leading to a number of proposals of mechanisms with novel features (3-7). However, as more results accumulated for this class of enzymes, possibilities for the mechanism became increasingly limited as cofactors and catalytic groups continued to be excluded from consideration (8-10). The high-resolution x-ray structure of ODCase from Methanobacterium thermoautotrophicum reveals that the mechanism is almost fully characterized by the formal description, along with electrostatic features of the enzyme's active site that provide selective destabilization of the orotidine group. In what follows, we report the results from a joint experimental and theoretical investigation, providing a mechanism that involves significant ground state destabilization effects in enzyme catalysis (11).The key to ODCase's catalytic power is its ability to utilize a phenomenon, which we classify as electrostatic stress [following Fersht's description of ''stress'' in catalysis (12)]. Although binding of the orotidine 5Ј-monophosphate (OMP) results in significant stabilizing interactions with the phosphate and ribose in the active site as revealed by the x-ray structural analysis, electrostatic interactions between the orotate group and ODCase is strongl...

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

“…Because development of the O4 anion resonance structure is essential for stabilization of a carbene-like intermediate, the relatively small stabilization effects by the Ser-127 backbone amide suggest that it is unlikely to be a major resonance form for the decarboxylation intermediate. Therefore, in the context of the 10 17 rate enhancement (23-kcal/mol decrease in ΔG ‡ ), the interaction between the backbone amide and O4 provides one of several modest contributions; others include substrate destabilization by enforced proximity of the substrate carboxylate group to Asp-70 in an otherwise hydrophobic pocket (5,8).…”

“…The rate enhancement is a composite of substrate destabilization and intermediate stabilization (Fig. 1A) (4)(5)(6)(7)(8)(9)(10)(11). The pK a of the UMP product is reduced from 30 to 34 in solution to ≤22 in the active site, requiring significant stabilization of the vinyl anion intermediate (∼14 kcal/mol) (12)(13)(14).…”

An Integrated Path Integral and Free-Energy Perturbation−Umbrella Sampling Method for Computing Kinetic Isotope Effects of Chemical Reactions in Solution and in Enzymes

“…46 As in numerous other decarboxylation reactions, the AM1 method yielded excellent energetic results in comparison with high-level electronic structural results. [47][48][49][50][51] In the case of the deprotonation of nitroethane by acetate ions, the AM1 model…”

“…This is in contrast to the decarboxylation of N-methyl orotate in water, which has little solvent effect. 47 The difference is again due to the presence of the positive charge in the pyridine ring, which is annihilated in the decarboxylation reaction. However, the similarity is that there is a small solvent reorganization barrier for CO 2 recombination, which is about 2 kcal/ mol.…”

Simulating enzyme reactions: Challenges and perspectives

“…131 This scheme has been supported by simulations with a semiempirical QM/MM hybrid potential that determined the freeenergy profiles for the enzyme and solution-phase reactions and the free energy of transfer of the substrate between enzyme and solution. 117 .…”