UC Riverside

This thesis presents progress in the adoption of NMR crystallography – the synergistic combination of X-ray crystallography, NMR spectroscopy, and Ab initio computational approaches – to study structures and dynamics of enzymes. The objective is to develop exceptionally definite and chemically-detailed structures of the chemical active sites by focusing on the intermediates along the pyridoxal-5'-phosphate catalyzed reaction pathway of tryptophan synthase.

NMR can supply direct chemical shifts to certain selected atoms in enzyme-substrate complexes; however, it is extremely sensitive to the local environment, making large complexes, such as enzyme complexes, unable to be interpreted. When the NMR shifts combine with X-ray crystallography, they can provide the framework to build computational models of active sites. The results of Ab initio calculations can reveal the unprecedented level of structural details. This is addressed in particular by the ketoenamine and enolimine tautomerization, in which a proton transfer points to the importance of protonation/deprotonation at ionizable sites on the coenzyme, substrates, and side residues to activate key steps in the catalytic process.

Solid-state NMR suffers from low sensitivity due to low polarization and slow polarization recovery times. With aid of dynamic nuclear polarization at low temperatures, solid-state NMR can not only enhance sensitivity but also capture some kinetic intermediates. These intermediates can be tautomers if solid-state NMR with dynamic nuclear polarization can selectively stabilize a tautomer. The use of NMR crystallography, which builds models for tautomers, provides a holistic view of the protonation states and dynamics of tautomers.