title: Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution.
abstract: 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 (∼10(17)) 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 ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). 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 (∼10(2)) to the 10(17) 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.
authors.affiliation: Departments of Biochemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801;
authors.forename: Bijoy J
authors.initials: BJ
authors.lastname: Desai
authors.affiliation: Department of Chemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan;
authors.forename: Yuki
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455;
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cembran
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; and.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Fedorov
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; and.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455; State Key Laboratory of Theoretical and Computational Chemistry, Jilin University, Changchun 130061, Jilin Province, People's Republic of China.
authors.forename: Jiali
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Chemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan;
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suga
authors.affiliation: Departments of Biochemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; Chemistry and j-gerlt@uiuc.edu.
authors.forename: John A
authors.initials: JA
authors.lastname: Gerlt
mesh_terms: D000577:Amides / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002474:Cell-Free System; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000737:chemistry*; D019605:Euryarchaeota / Q000201:enzymology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry*; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D000577:Amides; D004952:Esters; D016337:RNA, Catalytic; D012343:RNA, Transfer; D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: cell-free translation; enzymology; flexible tRNA acylation ribozyme; unnatural protein residue