The structural and biochemical impacts of monomerizing human acetylcholinesterase
Date
2019Journal
Protein SciencePublisher
Blackwell Publishing LtdType
Article
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Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit–subunit interactions on the structure of hAChE, a 2.1 Å X‐ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S) − VX (ethyl({2‐[bis(propan‐2‐yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI‐6 (1‐(2′‐hydroxyiminomethyl‐1′‐pyridinium)‐3‐(4′‐carbamoyl‐1‐pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site. Copyright 2019 The Authors.Sponsors
This work was funded by the Defense Threat Reduction Agency project: CB#3889 ?Elucidation of the mechanisms and physical properties of the molecular targets of chemical nerve agents? (JJH & SDP).Identifier to cite or link to this item
https://www.scopus.com/inward/record.uri?eid=2-s2.0-85065438023&doi=10.1002%2fpro.3625&partnerID=40&md5=36693c2c6f993501b831694f57d94f92; http://hdl.handle.net/10713/10798ae974a485f413a2113503eed53cd6c53
10.1002/pro.3625
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