Dataset Information

A model of glycosylated human butyrylcholinesterase.

ABSTRACT: Human butyrylcholinesterase (BChE) and its mutants have shown great potential in treating cocaine overdose and addiction. In order to effectively suppress cocaine reward in the brain for a long period of time after an exogenous cocaine hydrolase administration, the therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long circulation time. It has been known that PEGylation (covalent attachment of polyethylene glycol) modification of a therapeutic protein can prolong the biological half-life of the protein without affecting its biological function. However, the asparagine-linked glycans on the surface of glycosylated BChE may interfere with the PEGylation modification. In this study, we built a three-dimensional (3D) model of glycosylated human BChE to investigate the influence of glycans on the PEGylation modification. Glycans did not change the overall stability of the BChE structure, but could increase the flexibility of some local structures. For further evaluating the accessibility of the PEGylation reaction sites, particularly lysine residues, on the protein surface, we calculated the Solvent Accessible Surface Areas (SASAs) of these residues. The results indicate that some lysine residues show a significant decrease in SASA due to the direct or indirect influence of their surrounding glycans. The results also indicate that PEGylation reaction agents with smaller functional groups could have a better chance to react with lysine residues. This investigation provides a structural basis for rational engineering of human BChE and its mutants as therapeutic candidates.

SUBMITTER: Fang L

PROVIDER: S-EPMC3947857 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.

Dataset Information

A model of glycosylated human butyrylcholinesterase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets