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Investigation of Human Serine Racemase Mechanism and Development and Deployment of a Sensitive, Information-Rich Inhibitor Screen
Human Serine Racemase (hSR) is a vitamin B6-dependent enzyme that is responsible for the synthesis of N-methyl-D-aspartate receptor (NMDAr) co-agonist D-serine. Binding at the glycine site of the NMDAr with co-agonist L-glutamate allows for calcium influx in a critical neuronal signal event. While normal homeostatic function of hSR and the NMDAr are essential for proper learning and memory function (long term potentiation), elevation of D-serine has been associated with ischemic stroke, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Hypofunction of the enzyme is associated with schizophrenia. Additionally, aside from the racemization of LD-serine hSR can β-eliminate water from L-serine, producing pyruvate. The pyruvate production has been recently associated with colorectal cancer by way of histone H-3 acetylation. Chapter 1 provides an in-depth analysis of pyridoxal-5’-phosphate (PLP) dependent enzymes. A focus is taken on the mechanistic interplay of serine racemase residues and on understanding their importance to enzymatic function. Chapter 2 demonstrates the development of a novel dual function assay to monitor both hSR functions, -deprotonation/reprotonation and -elimination to pyruvate, in parallel. The NMR assay utilizes segregated 13C-labeling at C-2 and C-3 to enhance signal and exploits 13C/2H crosstalk, specifically the shielding/splitting effects of deuterium upon the 13C-signals. A screen of 1020 compounds with this assay revealed a dynamic range of three orders of magnitude and allowed for the identification of several new inhibitor chemotypes. Structure-activity-relationship (SAR) studies were performed on the most promising, indolo-chroman-2,4-dione family. Exhaustive kinetic analysis shows non-competitive inhibition, suggestive of binding to the allosteric, adenosine-triphosphate (ATP) binding pocket. The assay reveals the previously undetectable si-face proton-exchange reaction, now the most sensitive measure of hSR active site chemistry with the native substrate. The assay also provides for the indirect observation of the putative, carbanionic intermediate and yields a quantitative picture of the tripartite partitioning of this intermediate. Chapter 3 provides an overview of the nature of the key carbanionic intermediate-localized vs. delocalized quinonoid-in
Graham, Danielle, "Investigation of Human Serine Racemase Mechanism and Development and Deployment of a Sensitive, Information-Rich Inhibitor Screen" (2022). ETD collection for University of Nebraska - Lincoln. AAI29208470.