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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.