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An Integrated Study of PLP-Dependent Enzyme Mechanisms Through Targeted Mutagenesis, Inhibitor Design and Kinetic Evaluation
The research in Chapters 1 and 2 focuses on developing inactivators of pyridoxal-5’-phosphate (PLP) enzymes. The work within Chapter 1 contributes to a methodology that facilitates the introduction of a methoxyvinyl group into a variety of systems with the future potential of developing PLP focused inactivators inspired by the natural product methoxyvinylglycine. The work within Chapter 2 describes the first synthesis of b,b-difluorovinyl phenyl sulfone, a highly sought after, but elusive, electrophile that we demonstrate also serves as a (1’-fluoro)vinyl cation equivalent. This electrophile is utilized to synthesize quaternary α-(1’-fluoro)vinyl amino acids, bearing the native side chains of eight amino acids. The α-(1’-fluoro)vinyl analogue of lysine is shown to irreversibly inactivate the model enzyme lysine decarboxylase. This new inhibitor class has the potential to target a large variety of PLP-dependent enzymes. Chapter 3 turns to studies directed at developing inhibitors for an important PLP enzyme, cystathionine β-synthase (CBS). CBS is a β-eliminase/replacement enzyme that catalyzes L,L-cystathionine biosynthesis, a key step in the transulfuration pathway. CBS is also responsible for the production of H2S as a “gaseous hormone” in the brain. CBS overexpression pursuant to ischemic stroke appears to be a key element leading to neuronal cell damage. The focus here was to synthesize C2-symmetric cystathionine-based mimics to effectively inhibit the enzyme and test for increased cell viability in stroke model systems. A fully saturated hydrazino-mimic showed the strongest inhibition of the compounds tested in vitro. This newly found inhibitor led to ~ 70% reduction of infarct volume upon ICV administration 1 h post-occlusion in a rat model for stroke. Chapter 4 focuses on another enzyme associated with neuronal signaling, serine racemase, which produces D-serine, a co-agonist of the NMDA receptor associated with learning and memory. Site-directed mutagenesis of the active site re-face base (S84D, S84N, S84T) coupled with steady enzyme kinetics on a set of substrates and inhibitors revealed dramatic changes in substrate specificity seen as a function of re-face base. Molecular modeling and docking allowed one to rationalize these effects and suggests key roles for substrate positioning and stereoelectronics in this active site.
Beio, Matthew L, "An Integrated Study of PLP-Dependent Enzyme Mechanisms Through Targeted Mutagenesis, Inhibitor Design and Kinetic Evaluation" (2017). ETD collection for University of Nebraska - Lincoln. AAI10683823.