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

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.

Fig 1. Purification of human carboxylesterases.

(a) Size exclusion profiles of purified hCES1 (blue trace) hCES1 N79Q (green trace) and hCES1 S221A (red trace) enzymes from media of transfected HEK cells. Samples were run on a HiLoad 16/60Superdex 200 column (GE Healthcare) in 200 mM NaCl, 20 mM Tris-HCl, pH 7.5. The peak corresponds to a molecular weight of approximately 160 kDa as estimated from the elution volumes of globular proteins of known molecular weight: Aprotinin (6.5 kDa) Ribonuclease A (13.7 kDa) Carbonic Anhydrase (29 kDa), Ovalbumin (44 kDa), Conalbumin (75 kDa) Aldolase (158 kDa) Ferritin (440 kDa) and Blue Dextran 2000. (b) SDS-polyacrylamide gel of purified CES1 (lanes 1 and 2) and CES1 N79Q (lanes 3 and 4) untreated (lanes 1 and 3) and treated with PNGaseF (lanes 2 and 4). (c) The sedimentation velocity distribution for hCES1 N79Q. Data for hCES1 and hCES1 S221A gave the same profiles.

Fig 2. Enzyme activity of human carboxylesterases.

Plots of initial rates of reaction against enzyme concentration assayed as described in the Methods section at a substrate concentration of 750 μM 4-NPA (a) CES1 (b) CES1 N79Q. Plots of initial reaction rates against substrate concentration for the hydrolysis of 4-NPA by (c) hCES1 and (d) hCES1 N79Q (3.4 nM each enzyme). The molarity of the enzyme was calculated assuming 100% trimer with a molecular weight of 182.7 kDa.

Fig 3. Crystal structures of human carboxylesterases.

(a) Overlay of hCES1 (blue) and hCES1 N79Q (gold) (b) Views of the glycosylation site for hCES1, S122A hCES1 and the N79Q mutant. The Fo–Fc omit map electron density is shown carved around the glycosylation site (N79) and contoured at a level of 3 σ (c) Trimer of hCES1 generated by the space group symmetry, N-acetylglucosamine sugars attached to N97 of hCES1 are represented as spheres in red and green (d) Superpostion of the catalytic triad of hCES1 (mauve), hCES1 N79Q (green), hCES1 S221A (yellow) hCES1 tamoxifen complex (PDB id 1ya4, turquoise). Residues are in stick representation. The bound tamoxifen is coloured turquoise. (e) Cartoon representation of the hCES1 trimer generated by the space group symmetry with the K78:E183 and K275:E292 salt bridges shown in spheres (K78 in green, E183 in magenta, K275 in cyan and E292 in orange).