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

Significance Orotidine 5′-monophosphate decarboxylase has attracted intense enzymological interest, because it achieves a very large rate enhancement (∼10 17 ) without the use of cofactors. Previous studies provided evidence that substrate destabilization and vinyl anion intermediate stabilization contribute to the rate enhancement. Using in vitro translation, we generated a backbone amide to ester substitution to evaluate the importance of the hydrogen bond between a backbone amide and the substrate in intermediate stabilization. The hydrogen bond contributes modestly (≤10 2 ), suggesting that the intermediate is primarily stabilized by electrostatic interactions with the active site. This study establishes a versatile method for generation of backbone amide to ester substitutions in sufficient quantities to investigate the importance of backbone amide hydrogen bonding interactions in enzyme-catalyzed reactions.