Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1
Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generat...
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| Format: | Article |
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Public Library of Science
2015
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| Online Access: | https://eprints.nottingham.ac.uk/41961/ |
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| author | Arena de Souza, Victoria Scott, David J. Nettleship, Joanne E. Rahman, Nahid Charlton, Michael H. Walsh, Martin A. Owens, Raymond J. |
| author_facet | Arena de Souza, Victoria Scott, David J. Nettleship, Joanne E. Rahman, Nahid Charlton, Michael H. Walsh, Martin A. Owens, Raymond J. |
| author_sort | Arena de Souza, Victoria |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme. |
| first_indexed | 2025-11-14T19:47:13Z |
| format | Article |
| id | nottingham-41961 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:47:13Z |
| publishDate | 2015 |
| publisher | Public Library of Science |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-419612020-05-04T17:27:00Z https://eprints.nottingham.ac.uk/41961/ Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1 Arena de Souza, Victoria Scott, David J. Nettleship, Joanne E. Rahman, Nahid Charlton, Michael H. Walsh, Martin A. Owens, Raymond J. Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme. Public Library of Science 2015-12-11 Article PeerReviewed Arena de Souza, Victoria, Scott, David J., Nettleship, Joanne E., Rahman, Nahid, Charlton, Michael H., Walsh, Martin A. and Owens, Raymond J. (2015) Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1. PLoS ONE, 10 (12). e0143919/1-e0143919/12. ISSN 1932-6203 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143919 doi:10.1371/journal.pone.0143919 doi:10.1371/journal.pone.0143919 |
| spellingShingle | Arena de Souza, Victoria Scott, David J. Nettleship, Joanne E. Rahman, Nahid Charlton, Michael H. Walsh, Martin A. Owens, Raymond J. Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1 |
| title | Comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| title_full | Comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| title_fullStr | Comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| title_full_unstemmed | Comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| title_short | Comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| title_sort | comparison of the structure and activity of
glycosylated and asglycosylated human carboxylesterase 1 |
| url | https://eprints.nottingham.ac.uk/41961/ https://eprints.nottingham.ac.uk/41961/ https://eprints.nottingham.ac.uk/41961/ |