The modification of nuclear and cytoplasmic protein with O-linked N-acetylglucosamine (O-GlcNAc) residues is a unique cellular process in higher eukaryotes. Levels of this modification are regulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine: polypeptidyl transferase (OGT) and O-GlcNAcase (OGA). Based on structural information obtained for an OGT homolog from Xanthomonas campestris, we characterized the active center of human OGT and proposed histidine 558 to be the catalytic general base. We also described a method to generate O-GlcNAcylated recombinant proteins in E. coli. This allowed, for the first time, kinetic studues of human OGA to be carried out using protein substrates. In combination with human OGT kinetics on the same substrates, we are able to predicted the relative O-GlcNAc stoichiometry between the protein substrates. Lastly, we explored the substrate specificity of human OGT and discovered uridine diphosphate glucose (UDP-Glc) may be a substrate of OGT within cells.
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Thesis advisor: Vocadlo, David
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