Theoretical Chemistry Accounts

, Volume 125, Issue 3–6, pp 375–386 | Cite as

Rationalizing perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular dynamics simulations of the second tetrahedral intermediate state

  • Wook Lee
  • Ljubica Vojcic
  • Dragana Despotovic
  • Radivoje Prodanovic
  • Karl-Heinz Maurer
  • Ulrich Schwaneberg
  • Martin ZachariasEmail author
Regular Article


The perhydrolysis reaction in hydrolases is an important example of catalytic promiscuity and has many potential industrial applications. The mechanisms of perhydrolase activity of a subtilisin Carlsberg mutant and of an aryl-esterase mutant have been investigated using classical molecular dynamics simulations of the second tetrahedral intermediate (TI) state. The simulations demonstrated that hydrogen bonding between the second TI of the perhydrolysis reaction is possible in the mutants but not wild type. The stabilization by hydrogen bonds was specific for the perhydrolysis intermediate and either no hydrogen bonding or only weakened hydrogen bonding to the second TI state of the hydrolysis reaction was observed. Furthermore, a significant hindrance to the formation of the catalytically important hydrogen bond between His64 and Ser221 in the catalytic triad by competing hydrogen bonds was found for the subtilisin mutant but not wild type enzyme in case of the hydrolysis intermediate. The opposite was observed in case of the perhydrolysis intermediate. The result offers a qualitative explanation for the overall reduced hydrolysis activity of the subtilisin mutant. In addition, the simulations also explain qualitatively the perhydrolysis activity of the enzyme variants and may be helpful for designing enzyme mutants with further improved perhydrolysis activity.


Molecular simulation Enzyme promiscuity Perhydrolysis catalysis Intermediate stabilization 



This work was performed using the computational resources of the CLAMV (Computer Laboratories for Animation, Modeling and Visualization) at Jacobs University Bremen and supercomputer resources of the EMSL (Environmental Molecular Science Laboratories) at the PNNL (Pacific Northwest National Laboratories).

Supplementary material

214_2009_611_MOESM_ESM.doc (115 kb)
Supplementary material (DOC 115 kb)


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Wook Lee
    • 2
  • Ljubica Vojcic
    • 1
  • Dragana Despotovic
    • 1
  • Radivoje Prodanovic
    • 1
  • Karl-Heinz Maurer
    • 3
  • Ulrich Schwaneberg
    • 4
  • Martin Zacharias
    • 1
    • 5
    Email author
  1. 1.School of Engineering and ScienceJacobs UniversityBremenGermany
  2. 2.Institute of Organic ChemistryUniversity of WürzburgAm HublandWürzburg
  3. 3.WRC-Biotechnology, Henkel AG & Co. KGaADüsseldorfGermany
  4. 4.RWTH AachenWorringerweg 1AachenGermany
  5. 5.Physics DepartmentTechnical University MunichGarchingGermany

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