Chemical probing of glycogenesis
<p>The polysaccharide molecule glycogen represents a crucial glucose (and hence energy) storage reserve present in a wide variety of organisms. In eukaryotes, its biosynthesis is initiated by the glycosyltransferase enzyme Glycogenin, which unusually catalyses its own autoglucosylation to form...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2018
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| _version_ | 1797090390266871808 |
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| author | Bilyard, M |
| author2 | Davis, B |
| author_facet | Davis, B Bilyard, M |
| author_sort | Bilyard, M |
| collection | OXFORD |
| description | <p>The polysaccharide molecule glycogen represents a crucial glucose (and hence energy) storage reserve present in a wide variety of organisms. In eukaryotes, its biosynthesis is initiated by the glycosyltransferase enzyme Glycogenin, which unusually catalyses its own autoglucosylation to form a short maltosaccharide extending from a tyrosine residue. Glycogenin’s privileged position at the heart of carbohydrate metabolism has sparked wide interest in elucidating its catalytic mechanism. However, prior studies have been hindered by an inability to access homogeneous glycoforms of the enzyme, restricting precise mechanistic analyses.</p> <p>Suzuki-Miyaura cross-coupling represents a widely utilised reaction in organic chemistry and has more recently emerged as a powerful tool for the site-selective modification of proteins. In this thesis, we present the application of this methodology to the mechanistic study of Glycogenin, utilising a “genetically tagged” variant of the latter bearing an unnatural p-iodophenylalanine residue in place of the catalytic tyrosine. Preliminary studies demonstrate the potential of this enzyme to undergo efficient cross-coupling under benign, “enzyme-compatible” conditions. This leads to the application of this reaction as part of a novel palladium-mediated enzyme activation process for Glycogenin, allowing unprecedented access to different, homogeneously glucosylated enzyme states. These closely mimic intermediates at different stages of the autoglucosylation pathway, and are therefore catalytically active. </p> <p>Comparative kinetic analyses of such Glycogenin constructs reveal unusual triphasic kinetics for the initiation of glycogen biosynthesis. Subsequent studies on both a non-natural autogalactosylation pathway and on autoglucosylation pathways initiating from non-glucose carbohydrate glycosyl acceptors reveal a surprising degree of enzyme plasticity towards glycosyl donor and acceptor, and suggest that the three kinetic phases may be additionally defined in this regard.</p> <p>We also apply our methodology to investigation of a Glycogenin mutation implicated in a pathological condition arising from impaired autoglucosylation. Comparison of previously inaccessible glycoforms of this mutant challenges prior beliefs that it is entirely incapable of autoglucosylation and highlights further differences between early catalytic phases of catalysis. We anticipate that our approach may in the future enable mechanistic investigation of the other metabolic disorders and neurodegenerative conditions associated with malfunctions in glycogen biosynthesis.</p> |
| first_indexed | 2024-03-07T03:17:53Z |
| format | Thesis |
| id | oxford-uuid:b66bd3aa-b195-4dcc-b55f-4e20cdf527eb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:17:53Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | oxford-uuid:b66bd3aa-b195-4dcc-b55f-4e20cdf527eb2022-03-27T04:40:48ZChemical probing of glycogenesisThesishttp://purl.org/coar/resource_type/c_db06uuid:b66bd3aa-b195-4dcc-b55f-4e20cdf527ebEnglishORA Deposit2018Bilyard, MDavis, B<p>The polysaccharide molecule glycogen represents a crucial glucose (and hence energy) storage reserve present in a wide variety of organisms. In eukaryotes, its biosynthesis is initiated by the glycosyltransferase enzyme Glycogenin, which unusually catalyses its own autoglucosylation to form a short maltosaccharide extending from a tyrosine residue. Glycogenin’s privileged position at the heart of carbohydrate metabolism has sparked wide interest in elucidating its catalytic mechanism. However, prior studies have been hindered by an inability to access homogeneous glycoforms of the enzyme, restricting precise mechanistic analyses.</p> <p>Suzuki-Miyaura cross-coupling represents a widely utilised reaction in organic chemistry and has more recently emerged as a powerful tool for the site-selective modification of proteins. In this thesis, we present the application of this methodology to the mechanistic study of Glycogenin, utilising a “genetically tagged” variant of the latter bearing an unnatural p-iodophenylalanine residue in place of the catalytic tyrosine. Preliminary studies demonstrate the potential of this enzyme to undergo efficient cross-coupling under benign, “enzyme-compatible” conditions. This leads to the application of this reaction as part of a novel palladium-mediated enzyme activation process for Glycogenin, allowing unprecedented access to different, homogeneously glucosylated enzyme states. These closely mimic intermediates at different stages of the autoglucosylation pathway, and are therefore catalytically active. </p> <p>Comparative kinetic analyses of such Glycogenin constructs reveal unusual triphasic kinetics for the initiation of glycogen biosynthesis. Subsequent studies on both a non-natural autogalactosylation pathway and on autoglucosylation pathways initiating from non-glucose carbohydrate glycosyl acceptors reveal a surprising degree of enzyme plasticity towards glycosyl donor and acceptor, and suggest that the three kinetic phases may be additionally defined in this regard.</p> <p>We also apply our methodology to investigation of a Glycogenin mutation implicated in a pathological condition arising from impaired autoglucosylation. Comparison of previously inaccessible glycoforms of this mutant challenges prior beliefs that it is entirely incapable of autoglucosylation and highlights further differences between early catalytic phases of catalysis. We anticipate that our approach may in the future enable mechanistic investigation of the other metabolic disorders and neurodegenerative conditions associated with malfunctions in glycogen biosynthesis.</p> |
| spellingShingle | Bilyard, M Chemical probing of glycogenesis |
| title | Chemical probing of glycogenesis |
| title_full | Chemical probing of glycogenesis |
| title_fullStr | Chemical probing of glycogenesis |
| title_full_unstemmed | Chemical probing of glycogenesis |
| title_short | Chemical probing of glycogenesis |
| title_sort | chemical probing of glycogenesis |
| work_keys_str_mv | AT bilyardm chemicalprobingofglycogenesis |