UC Riverside

This dissertation aims to provide a full mechanistic understanding of enzyme catalysis by investigating the active site protonation states and chemical dynamics. Accurate knowledge of proton positions and sidechain orientations is crucial for establishing the mechanism of acid-base catalysis and allostery in enzymes These finite details are necessary for developing various computational techniques such as molecular dynamics simulations, molecular docking routines, and structure-based drug design. While high-resolution X-ray crystal structures provide information about protein residues and cofactors interacting with the substrate, they often fail to determine protonation states due to insufficient resolution. Currently, neutron crystallography is making strides in defining heavy and hydrogen atom locations but is limited by the need for large, preferably perdeuterated, crystals and multi-week-long acquisition times. Cryo-EM is also making progress in hydrogen atom detection but is far from routine.Nuclear magnetic resonance (NMR) spectroscopy has been proven to be a sensitive tool for detecting chemical environment in enzyme active sites and can be combined with Cryo-EM and diffraction methods for atomic-resolution descriptions of structure and function. However, to delineate the chemistry of the active site, NMR and diffraction are more powerful when combined with first-principles computational chemistry. This dissertation presents a comprehensive investigation of enzyme catalysis by combining X-ray Crystallography, NMR Spectroscopy and First Principles Calculations in a synergistic approach of NMR Crystallography to determine enzyme mechanism and establish a full understanding of the catalytic pathway.