dc.contributor.author | McGary, Laura | |
dc.date.accessioned | 2022-08-19T12:52:45Z | |
dc.date.available | 2022-08-19T12:52:45Z | |
dc.date.issued | 2022-08-19 | |
dc.identifier.uri | http://hdl.handle.net/10222/81860 | |
dc.description | In this thesis, three major topics will be discussed: (i) investigations of protein architectures using an activity-based probe, (ii) evaluation of the inactivation of L-fuconate dehydratase by 3-hydroxypyruvate and the effects of Tris buffer, and (iii) the re-engineering of glutamate racemase to address the question of whether the enzyme can be made preferentially “unidirectional”. | en_US |
dc.description.abstract | Sodium methyl hex-5-ynoyl phosphate (SMHP) was synthesized as an activity-based probe designed to target protein architectures with nucleophiles adjacent to a cationic binding site. Activity-based protein profiling revealed that SMHP modified 281 enzymes from the cell-lysate of Pseudomonas lemognei, including D-3-hydroxybutyrate dehydrogenase and CTP synthase. The sites of modification were investigated using fluorescence-based activity studies, LC-MS/MS, and kinetics. The inactivation of L-fuconate dehydratase (FucD) by 3-hydroxypyruvate and the effects of Tris buffer were also explored. With increasing Tris concentrations, the kinactapp and KIapp values decreased, but the kinactapp/KIapp remained unchanged (~0.018 ± 0.002 M–1s–1). Finally, the Cys to Ser variants (C76S and C186S) of glutamate racemase from Fusobacterium nucleatum were constructed. The C76S variant exhibited greater catalytic efficiency turning over D-Glu relative to L-Glu at higher pH values; however, this preferred ‘unidirectional’ behavior was not observed for the C186S variant in the L-Glu to D-Glu reaction direction. | en_US |
dc.language.iso | en | en_US |
dc.subject | protein architectures | en_US |
dc.subject | activity-based probe | en_US |
dc.subject | enzymes | en_US |
dc.title | Investigations of enzyme active-site architectures and catalysis | en_US |
dc.type | Thesis | en_US |
dc.date.defence | 2022-08-12 | |
dc.contributor.department | Department of Biochemistry & Molecular Biology | en_US |
dc.contributor.degree | Master of Science | en_US |
dc.contributor.external-examiner | n/a | en_US |
dc.contributor.graduate-coordinator | Dr. Jamie Kramer | en_US |
dc.contributor.thesis-reader | Dr. Jan K. Rainey | en_US |
dc.contributor.thesis-reader | Dr. David L. Jakeman | en_US |
dc.contributor.thesis-reader | Dr. K. Vanya Ewart | en_US |
dc.contributor.thesis-supervisor | Dr. Stephen L. Bearne | en_US |
dc.contributor.ethics-approval | Not Applicable | en_US |
dc.contributor.manuscripts | Not Applicable | en_US |
dc.contributor.copyright-release | Not Applicable | en_US |