title: NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.
abstract: Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cαand positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Bethany G
authors: BG
authors: Caulkins
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Robert P
authors: RP
authors: Young
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Ryan A
authors: RA
authors: Kudla
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Chen
authors: C
authors: Yang
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Thomas J
authors: TJ
authors: Bittbauer
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Baback
authors: B
authors: Bastin
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Eduardo
authors: E
authors: Hilario
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Li
authors: L
authors: Fan
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Michael J
authors: MJ
authors: Marsella
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Michael F
authors: MF
authors: Dunn
authors: Department of Chemistry, and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors: Leonard J
authors: LJ
authors: Mueller
mesh_terms: D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011789:Quantum Theory; D012486:Salmonella typhimurium / Q000201:enzymology; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D014367:Tryptophan Synthase