期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：42
页码：15066-15071
DOI：10.1073/pnas.1411772111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceOrotidine 5'-monophosphate decarboxylase has attracted intense enzymological interest, because it achieves a very large rate enhancement ([~]1017) 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 ([≤]102), 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. Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement ([~]1017) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate (HOS) was used to generate the ester backbone substitution (S127HOS). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by [~]1.5-fold and decreased the value of kcat by [~]50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor ([~]102) to the 1017 rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.
