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Comparison of the structure and activity of glycosylated and aglycosylated human carboxylesterase 1

DOI: 10.1371/journal.pone.0143919 DOI Help
PMID: 26657071 PMID Help

Authors: Victoria Arena De Souza (The Research Complex at Harwell) , David J. Scott (The Research Complex at Harwell) , Joanne E. Nettleship (The Research Complex at Harwell) , Nahid Rahman (The Research Complex at Harwell) , Michael H. Charlton (Chroma Therapeutics Ltd) , Martin Walsh (Diamond Light Source) , Raymond J. Owens (The Research Complex at Harwell)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos One , VOL 10

State: Published (Approved)
Published: December 2015

Open Access Open Access

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.

Journal Keywords: Crystal structure; Enzyme structure; Enzymes; Glycols; Glycosylation; Enzyme kinetics; Monomers; Enzyme purification

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Medicine

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 18/01/2016 11:19

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Discipline Tags:

Health & Wellbeing Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)