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Home / Equine Research Bank / Chem Biol Interact / A secondary isotope effect study of equine serum butyrylchol...
Chemico-biological interactions2010; 187(1-3); 124-127; doi: 10.1016/j.cbi.2010.05.007

A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

Abstract: beta-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L(3)-thiocholine (L=H or (2)H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis-Menten kinetics, and yield the following isotope effects: (D3)k(cat)/K(m)=0.98+/-0.02 and (D3)k(cat)=1.10+/-0.02. The modestly inverse isotope effect on k(cat)/K(m) is consistent with partial rate limitation by a step that converts the sp(2)-hybridized ester carbonyl of the E+A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k(cat) indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538-14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.
Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.
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Publication Date: 2010-05-20 PubMed ID: 20493178PubMed Central: PMC2912972DOI: 10.1016/j.cbi.2010.05.007Google Scholar: Lookup
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Summary

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The researchers of this study have examined equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine using beta-secondary deuterium isotope effects and have found that the rate of this process is controlled by the breakdown of a tetrahedral intermediate, a finding that correlates with and supports preceding research on acetylthiocholine hydrolysis.

Introduction

  • The study focuses on understanding the enzymatic reaction mechanism of acetylthiocholine (ATCh) hydrolysis catalyzed by equine serum butyrylcholinesterase, enzymes that play significant roles in nerve function and signal transmission.
  • This process was evaluated using beta-secondary deuterium isotope effects, a method used to provide insight into the nature of the transition states in chemical reactions.

Methodology

  • The researchers measured isotope effects for hydrolysis of acetyl-L(3)-thiocholine, where L is either hydrogen (H) or deuterium ((2)H).
  • They observed the dependencies of initial rates on isotopic substrate concentrations which was shown to closely follow Michaelis-Menten kinetics – a model that helps to understand the rate of enzyme-catalyzed reactions.

Findings

  • The isotope effects measured were (D3)k(cat)/K(m)=0.98+/-0.02, showing a slightly inverse effect, and (D3)k(cat)=1.10+/-0.02, displaying a distinctively normal isotope effect.
  • The modest inverse isotope effect on k(cat)/K(m) implies that the rate of the hydrolysis reaction is somewhat limited by the step that converts the ester carbonyl of the E+A reactant state into a quasi-tetrahedral transition state during the acylation stage of catalysis. “Acylation” refers to a chemical process where an acyl group is introduced to a molecule.
  • The normal isotope effect on k(cat) suggests that at substrate saturation, the breakdown of a tetrahedral intermediate that has accumulated at the active site during catalytic turnover is the rate-limiting step of the enzymatic process.

Conclusion

  • Overall, these outcomes support a previously reported study, according to which, the rate of substrate-activated hydrolysis of ATCh, catalyzed by recombinant human butyrylcholinesterase, is also limited by the breakdown of an accumulating tetrahedral intermediate.
  • Thus, this research enhances our understanding of enzyme-catalyzed reactions and specifically, the role of transition states in governing the rate at which these reactions progress.

Cite This Article

APA
Wiley KL, Tormos JR, Quinn DM. (2010). A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine. Chem Biol Interact, 187(1-3), 124-127. https://doi.org/10.1016/j.cbi.2010.05.007

Publication

Chemico-biological interactions
ISSN: 1872-7786
NlmUniqueID: 0227276
Country: Ireland
Language: English
Volume: 187
Issue: 1-3
Pages: 124-127

Researcher Affiliations

Wiley, Kenneth L
  • Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
Tormos, Jose R
    Quinn, Daniel M

      MeSH Terms

      • Acetylthiocholine / metabolism
      • Animals
      • Biocatalysis
      • Butyrylcholinesterase / blood
      • Butyrylcholinesterase / chemistry
      • Butyrylcholinesterase / metabolism
      • Crystallography, X-Ray
      • Deuterium
      • Horses
      • Humans
      • Hydrolysis
      • Kinetics

      Grant Funding

      • R21 NS076430 / NINDS NIH HHS
      • T32 GM008365 / NIGMS NIH HHS
      • T32 GM008365-19 / NIGMS NIH HHS
      • T32GM008365 / NIGMS NIH HHS

      References

      This article includes 13 references
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      Citations

      This article has been cited 3 times.
      1. Quinn DM, Topczewski J, Yasapala N, Lodge A. Why is Aged Acetylcholinesterase So Difficult to Reactivate?. Molecules 2017 Sep 4;22(9).
        doi: 10.3390/molecules22091464pubmed: 28869561google scholar: lookup
      2. Chen X, Fang L, Liu J, Zhan CG. Reaction pathway and free energy profiles for butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine. Biochemistry 2012 Feb 14;51(6):1297-305.
        doi: 10.1021/bi201786spubmed: 22304234google scholar: lookup
      3. Tormos JR, Wiley KL, Wang Y, Fournier D, Masson P, Nachon F, Quinn DM. Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study. J Am Chem Soc 2010 Dec 22;132(50):17751-9.
        doi: 10.1021/ja104496qpubmed: 21105647google scholar: lookup
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