Dataset Information

Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer-Based Protein Engineering.

ABSTRACT: Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of cholinesterase-organophosphate complexes, but suffer from short half-lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long-lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organophosphate nerve agents with one-to-one stoichiometries. The chemical power of nucleophilic reactivators is coupled to long-lived bioscavengers by designing and synthesizing cholinesterase-polymer-oxime conjugates using atom transfer radical polymerization and azide-alkyne "click" chemistry. Detailed kinetic studies show that butyrylcholinesterase-polymer-oxime activity is dependent on the electrostatic properties of the polymers and the amount of oxime within the conjugate. The covalent coupling of oxime-containing polymers to the surface of butyrylcholinesterase slows the rate of inactivation of paraoxon, a model nerve agent. Furthermore, when the enzyme is covalently inhibited by paraoxon, the covalently attached oxime induced inter- and intramolecular reactivation. Intramolecular reactivation will open the door to the generation of a new class of nerve agent scavengers that couple the speed and selectivity of biology to the ruggedness and simplicity of synthetic chemicals.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6947490 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer-Based Protein Engineering.

Zhang Libin L Baker Stefanie L SL Murata Hironobu H Harris Nicholas N Ji Weihang W Amitai Gabriel G Matyjaszewski Krzysztof K Russell Alan J AJ

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20191113 1

Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of cholinesterase-organophosphate complexes, but suffer from short half-lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long-lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organo ...[more]

PMID: 31921563

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Project description:The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, X-ray crystallography, and hydrogen-deuterium mass spectrometry, we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.

Dataset Information

Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer-Based Protein Engineering.

Publications

Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer-Based Protein Engineering.

Similar Datasets

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