UC Riverside

Understanding the dynamics of internal cavities of proteins large enough to host water molecules is an important step toward the analysis of the proton-transfer mechanism and water dynamics of PLP-dependent proteins. In the present study, the active site of the beta-subunit of Tryptophan Synthase was analyzed to determine the water molecule network present, its H-bond interactions with the amino acids as well as protonated states of cofactor and surrounding residues located in the α-aminoacrylate intermediate, and their roles in the proton-transfer mechanism. Here, we use method known as NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry. NMR crystallography offers unprecedented insight into three-dimensional, chemically-detailed structure in biomolecules and revealing chemically-rich detail concerning interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. We used NMR crystallography to create testable models for structure and function of enzyme active sites. Here, we employed this process to probe the active site in the β-subunit of tryptophan synthase with atomic-level resolution. NMR crystallography approach allows to perform cluster-based calculations to determine specific to the level of theory and basis sets magnetic shielding that can be converted later into the chemical shifts using rescaling parameters. The obtained calculated chemical shifts compared to the experimental values using chi-square statistical approach resulted in the detailed refinement of the structure and prediction of the proton exchange between tautomers. We determined that α-aminoacrylate intermediate formed during TS catalysis ensures a partial positive charge at Cβ of the serine substrate to facilitate nucleophilic attack by the incoming indole substrate. Reaction specificity is therefore accomplished through a combination of correct electrostatic environment (i.e. protonation states) and stereoelectronic control. The aminoacrylate structure also suggests the fate of the substrate’s β-hydroxyl group – it becomes the adjacent water molecule.