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

Abstract

The reaction catalyzed by orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for the rate enhancement [kcat/knon = 7.1 × 1016] and catalytic proficiency [(kcat/KM)/knon = 4.8 × 1022 M−1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that “substrate destabilization” by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ~5 kcal/mol (contributing ~2.7 × 103 to the rate enhancement) [K. K. Chan, B. M. Wood, A. A. Fedorov, E. V. Fedorov, H. J. Imker, T. L. Amyes, J. P. Richard, S. C. Almo, and J. A. Gerlt (2009) Biochemistry 48, 5518–31]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of kcat by as much as 400-fold but have minimal effect on the value of kex for exchange of H6 of the FUMP product analog with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions for Ser 127 that is proximal to O4 of the orotate ring decrease the value of kcat/KM, with the S127P substitution that eliminates hydrogen-bonding interactions with O4 producing a 2.5 × 106-fold reduction in the value of kcat/KM; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 to produce an anionic carbene intermediate and thereby provide a structural strategy for stabilization of the intermediate. These observations provide additional information on the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.

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).…”

“…Accordingly, two structural strategies have been proposed for stabilization of the anionic intermediate ( Fig. 1B): (i) an enhanced hydrogen-bonding interaction between the Ser-127 backbone amide and O4 of the pyrimidine moiety of the intermediate through a carbene resonance structure (anionic charge on O4) (8,11,15) and (ii) electrostatic interactions between the anionic charge on carbon-6 with the e-ammonium group of Lys-72 (4,9). Both are challenging to test: (i) substitutions for backbone amide groups are difficult to construct, and (ii) Lys-72 is the essential general acid catalyst.…”

“…We constructed the S127P mutant to evaluate the importance of the interaction of the backbone amide group with O4; the mutant has a dramatically reduced value for k cat /K M (10 6 -fold) compared with WT MtOMPDC. However, a comparison of liganded X-ray structures of the WT and S127P mutant revealed a displacement of BMP in the active site as the result of the steric interactions between the proline side chain and O4, thereby making interpretation of the importance of the interaction of the backbone amide group with O4 difficult (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).…”

Nuclear quantum effects and kinetic isotope effects in enzyme reactions

“…10 17 [151]. The catalytic mechanism of ODC has been subject to numerous investigations by both experimental [154][155][156][157][158][159][160][161][162][163][164] and computational [156,[165][166][167][168][169][170][171][172] means. In particular, Rishavy and Cleland employed 13 C and 15 N KIEs to elucidate the mechanism [164], and concluded that the most likely pathway involves a direct decarboxylation with an anionic intermediate [162,173].…”