Interactions of human butyrylcholinesterase with phenylvalerate and acetylthiocholine as substrates and inhibitors: kinetic and molecular modeling approaches

  • Jorge EstévezEmail author
  • Felipe Rodrigues de Souza
  • María Romo
  • Iris Mangas
  • Tanos Celmar Costa Franca
  • Eugenio Vilanova
Molecular Toxicology


Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site (“PV-site”). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.


Human butyrylcholinesterase Phenyl valerate Inhibition kinetics Docking Molecular dynamics 







Acetylcholinesterase (in general of any specie)


Butyrylcholinesterase (in general of any specie)




Cholinesterase (the activity)


Human butyrylcholinesterase


Molecular Mechanics Poisson Boltzman Solver Area


Neuropathy target esterase


Phenyl valerate


Phenyl valerate esterase (the activity)


Protein data bank



We are most grateful to Dr. Douglas Cerasoli [USAMRICD (US Army Medical Research Institute of Chemical Defense), Aberdeen Proving Ground, MD, USA] for the supply of hBChE.


Work was partly supported by project “Mecanismos moleculares de toxicidad de xenobióticos” from the University Miguel Hernández of Elche (Spain), (Reference; 50AV0050RP). This work was supported by the Brazilian financial agencies CAPES (fellowships), CNPq (Grant No 306156/2015-6), FAPERJ (Grant No E-02/202.961/2017), and also the by the excellence project FIM.

Supplementary material

204_2019_2423_MOESM1_ESM.docx (689 kb)
Supplementary material 1 (DOCX 689 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jorge Estévez
    • 1
    Email author
  • Felipe Rodrigues de Souza
    • 2
  • María Romo
    • 1
  • Iris Mangas
    • 1
    • 2
  • Tanos Celmar Costa Franca
    • 2
    • 3
  • Eugenio Vilanova
    • 1
  1. 1.Institute of BioengineeringUniversity Miguel HernándezElcheSpain
  2. 2.Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense (LMCBD)Military Institute of EngineeringRio de JaneiroBrazil
  3. 3.Center for Basic and Applied Research, Faculty of Informatics and ManagementUniversity of Hradec KraloveHradec KrálovéCzech Republic

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