NMR Crystallography in the Enzyme Active Site of Tryptophan Synthase

Saturday, 14 February 2015
Exhibit Hall (San Jose Convention Center)
Bethany G. Caulkins, UC Riverside, Riverside, CA
Background 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 cofactor in this extensive class of enzymes.  X-ray crystallography, optical spectroscopy, and physical-organic studies point to the importance of protonation/deprotonation at ionizable sites on the coenzyme, substrates, and sidechains to activate key steps in the catalytic process.  Yet direct characterization remains elusive as these techniques cannot specifically identify proton locations or report unambiguously on local chemical environment.  The chemical shift in nuclear magnetic resonance (NMR), however, is an extremely sensitive probe of chemical environment, but a large complex like a protein will give an enormous amount of data that can be inscrutable without guidelines for specific structure determination.  The use of computational chemistry aids in the creation of models that rely on specific chemical-level details and predicts detailed information like chemical shift.  Methods We employ NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry - to define three-dimensional, chemically-detailed structures of the intermediates in the tryptophan synthase cycle under conditions of active catalysis.  Together these methods can provide consistent and testable models for structure and function of enzyme active sites. Results Our results from studies on tryptophan synthase confirm some long-held mechanistic hypotheses, but also point to several novel structural hypotheses.  Support for the protonated Schiff base hypothesis, which postulates that the initial step in the catalytic cycle is facilitated by a protonated imine nitrogen, has been collected using 15N SSNMR, while evidence abounds that the carboxylate group of the substrate plays a larger role than previously supposed.  Conclusions For the internal aldimine, protonation of the Schiff-base linkage can be confirmed, while inspection of a nearby catalytic lysine sidechain residue shows a neutral amino group for the aminoacrylate complex.  For the quinonoid intermediate, a three-site proton exchange is suggested, with the acid site playing a larger role than previously anticipated.  We believe this has mechanistic significance as the acid isomer builds up negative charge at Cα, adding an electric field component along the reaction coordinate and lowering the energy barrier through charge stabilization.