The structural and biochemical impacts of monomerizing human acetylcholinesterase

Abstract

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.

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

“…So far, monomeric hAChE has not been observed in the crystalline state owing to high hAChE concentrations in the crystallization solutions, concentrations well above that of the dimer dissociation constant. The closest to the monomeric state was the X-ray structure of the Leu380Arg/Phe535Lys mutant of hAChE ( 9 ) that showed a severely distorted 4HB dimer ( Table S2 ). Analysis of our SAXS and cryo-EM data indicates that the 4HB homodimer is the predominant structure in solution at micromolar concentrations; thus, distortion of the 4HB interface would be expected to impair formation of homodimer.…”

“…In native AChE, catalytic properties of different molecular forms are generally assumed to be identical, in spite of slight variations in amino acid sequences at the C termini in some forms ( 6 , 7 ). Recombinant AChE forms, truncated at the C terminus to remove intersubunit -S-S- bonding in order to form monomeric AChE, are commonly used for structural studies and show catalytic properties identical to those of native AChE ( 7 , 8 , 9 , 10 ). No enzymatic cooperativity has been reported between subunits of oligomeric AChE ( 6 ).…”

“…Thus, there is the potential for considerable negative biological consequences associated with the dissociation of these 4HB dimers. A structural illustration on how destabilization in the dimeric structure at the 4HB interface could lead to nearly complete dimer dissociation in solution was reported for the Leu380Arg/Phe535Lys mutant of hAChE ( 9 ).…”

Design and Development of Cholinesterase Dual Inhibitors towards Alzheimer's Disease Treatment: A Focus on Recent Contributions from Computational and Theoretical Perspective

“…[7,23] The enzymes propel the breakdown of the neurotransmitter Ach into acetate and choline ( Figure 1) which result in the abortion of its signal. [24] AChEIs ( Figure 2) act by binding to the different sites buried at the base of a deep and narrow gorge lined by aromatic residues, and the peripheral anionic site (PAS) located at the gorge entry on the acetylcholinesterase. [26] For instance, donepezil acts by binding to the PAS of the AChE infusing both symptomatic effects in the AD treatment and causative ones holding back the deposition of amyloid plaque.…”

Reactivation of VX-Inhibited Human Acetylcholinesterase by Deprotonated Pralidoxime. A Complementary Quantum Mechanical Study

“…In our NAC, the distance between the O-atom of the aldoximate (−C=NO − ) group and the P-atom of the phosphorylated Ser203 (dOP) was 3.26 Å and the attack angle amongst the O-and P-atoms of the OP and the O-atom of Ser203 (OPO) was 175° [20]. These values, according to the NAC concept early defined by [20,21,25], resemble the bonds to be formed and broken in a pentacoordinate transition state (TS) [26][27][28] of a bimolecular nucleophile substitution (SN2) mechanism ( Figure 1) [29][30][31]. Experimental data for this reaction were published before by Nepovimova et al [32]; however, data about its energetics of reactivation are still missing.…”