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 its 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. 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. Our results from studies on tryptophan synthase confirm some long-held mechanistic hypotheses, but also point to several novel structural hypotheses.
|Advisor:||Mueller, Leonard J.|
|Commitee:||Bartels, Ludwig, Chronister, Eric L.|
|School:||University of California, Riverside|
|School Location:||United States -- California|
|Source:||DAI-B 79/02(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Physical chemistry|
|Keywords:||Nmr, Pyridoxal-5'-phosphate, Tryptohan synthase|
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