UC Riverside

Protonation and hybridization state are critical phenomena at the chemical-level that are vital in understanding an enzymes mechanism and function. Using high resolution X-ray crystallography unaccompanied is not enough to present a complete account of these details. On the other hand, the chemical shift from solid-state NMR spectroscopy is a remarkably insightful probe of the surrounding electro-chemical environment. The synergistic combination of NMR spectroscopy, X-ray crystallography and ab initio computational chemistry, also referred to as "NMR Crystallography", can be used as a unmatched tool for elucidating high resolution three-dimensional structures of diverse materials. This synergistic approach was used to determine the structures of the indoline-, 2-aminophenol (2AP)- quinonoid, and internal aldimine intermediates in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. In the presence of side-chain residues fixed at their crystallographically determined coordinates, models of the active site were created intending for the reactive substrate analogues to be optimized using ab initio computational chemistry. Experimentally measured chemical shifts at particular 13C - and 15N-labeled positions on the substrate are used to exclusively distinguish and emerge comparable species from the various computational model types; dictated by protonation state of the substrate analogues and nearby catalytic residues. The reduced chi squared analysis suggests the phenolic / phenolic-acid form to be the predominate protonated species for both the indoline and 2-aminophenol quinonoid intermediates, while the internal aldimine confirms the protonated Schiff base form.