title: Solution-State (17)O Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase.
abstract: Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Young
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Bethany G
authors.initials: BG
authors.lastname: Caulkins
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Borchardt
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Daryl N
authors.initials: DN
authors.lastname: Bulloch
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Cynthia K
authors.initials: CK
authors.lastname: Larive
authors.affiliation: Department of Biochemistry, University of California, Riverside, Riverside, CA, 92521, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
authors.affiliation: Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA. leonard.mueller@ucr.edu.
authors.forename: Leonard J
authors.identifier: http://orcid.org/0000-0002-2607-9875
authors.initials: LJ
authors.lastname: Mueller
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D014367:Tryptophan Synthase / Q000737:chemistry*
chemical_list: D014367:Tryptophan Synthase
keywords: NMR spectroscopy; enzymes; homogeneous catalysis; ligases; proteins