title: Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate.
abstract: The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.
authors.affiliation: Department of Chemistry, University of California , Riverside, California 92521, United States.
authors.forename: Bethany G
authors.initials: BG
authors.lastname: Caulkins
authors.forename: Baback
authors.initials: B
authors.lastname: Bastin
authors.forename: Chen
authors.initials: C
authors.lastname: Yang
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Neubauer
authors.forename: Robert P
authors.initials: RP
authors.lastname: Young
authors.forename: Eduardo
authors.initials: E
authors.lastname: Hilario
authors.forename: Yu-ming M
authors.initials: YM
authors.lastname: Huang
authors.forename: Chia-en A
authors.initials: CE
authors.lastname: Chang
authors.forename: Li
authors.initials: L
authors.lastname: Fan
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Marsella
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Mueller
mesh_terms: D020134:Catalytic Domain; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry* / Q000378:metabolism; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011522:Protons; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase