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1.
Chem Biol Interact ; 309: 108699, 2019 Aug 25.
Article in English | MEDLINE | ID: mdl-31202688

ABSTRACT

The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and intra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the α13 and α14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is ~10-8 s. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is -15.5 kcal/mol, while that for human AChE is -26.4 kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:1.


Subject(s)
Acetylcholinesterase/chemistry , Butyrylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Amino Acid Sequence , Butyrylcholinesterase/metabolism , Dimerization , Humans , Hydrophobic and Hydrophilic Interactions , Markov Chains , Molecular Dynamics Simulation , Protein Conformation, alpha-Helical , Sequence Alignment , Static Electricity , Thermodynamics
2.
Chem Biol Interact ; 306: 138-146, 2019 Jun 01.
Article in English | MEDLINE | ID: mdl-31009643

ABSTRACT

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 µs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.


Subject(s)
Biocatalysis , Butyrylcholinesterase/genetics , Butyrylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Computer-Aided Design , Echothiophate Iodide/pharmacology , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Butyrylcholinesterase/chemistry , Cholinesterase Inhibitors/chemistry , Echothiophate Iodide/chemistry , HEK293 Cells , Humans , Molecular Docking Simulation , Mutant Proteins/genetics , Quantum Theory
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