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Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry

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Abstract

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate—the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.

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Acknowledgments

This work was financially supported by the Australian Research Council, Discovery Projects Scheme (DP120104263). G.S. also acknowledges the receipt of an ARC Future Fellowship (FT120100694). M.P. is supported by an International Postgraduate Research Scholarship and University of Queensland International Living Allowance Scholarship.

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Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Brisbane, QLD, 4072, Australia

    Marcelo M. Pedroso, Fernanda Ely, Thierry Lonhienne, Lawrence R. Gahan, Luke W. Guddat & Gerhard Schenk

  2. Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia

    David L. Ollis

Authors
  1. Marcelo M. Pedroso
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  2. Fernanda Ely
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  3. Thierry Lonhienne
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  4. Lawrence R. Gahan
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  5. David L. Ollis
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  6. Luke W. Guddat
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  7. Gerhard Schenk
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Corresponding authors

Correspondence to Thierry Lonhienne or Gerhard Schenk.

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Pedroso, M.M., Ely, F., Lonhienne, T. et al. Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry. J Biol Inorg Chem 19, 389–398 (2014). https://doi.org/10.1007/s00775-013-1079-0

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  • DOI: https://doi.org/10.1007/s00775-013-1079-0

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