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Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Arena de Souza, Victoria; Scott, David J.; Nettleship, Joanne E.; Rahman, Nahid; Charlton, Michael H.; Walsh, Martin A.; Owens, Raymond J.

Authors

Victoria Arena de Souza

DAVID SCOTT DAVID.SCOTT@NOTTINGHAM.AC.UK
Associate Professor & Reader in Physical Biochemistry

Joanne E. Nettleship

Nahid Rahman

Michael H. Charlton

Martin A. Walsh

Raymond J. Owens



Contributors

Andreas Hofmann
Editor

Abstract

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.

Citation

Arena de Souza, V., Scott, D. J., Nettleship, J. E., Rahman, N., Charlton, M. H., Walsh, M. A., & Owens, R. J. (2015). Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1. PLoS ONE, 10(12), e0143919. https://doi.org/10.1371/journal.pone.0143919

Journal Article Type Article
Acceptance Date Nov 11, 2015
Online Publication Date Dec 11, 2015
Publication Date Dec 11, 2015
Deposit Date Apr 21, 2017
Publicly Available Date Mar 29, 2024
Journal PLOS ONE
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 10
Issue 12
Article Number e0143919
Pages e0143919
DOI https://doi.org/10.1371/journal.pone.0143919
Public URL https://nottingham-repository.worktribe.com/output/769432
Publisher URL http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143919

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