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

Pedroso, Marcelo M., Ely, Fernanda, Lonhienne, Thierry, Gahan, Lawrence R., Ollis, David L., Guddat, Luke W. and Schenk, Gerhard (2014) Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry. Journal of Biological Inorganic Chemistry, 19 3: 389-398. doi:10.1007/s00775-013-1079-0


Author Pedroso, Marcelo M.
Ely, Fernanda
Lonhienne, Thierry
Gahan, Lawrence R.
Ollis, David L.
Guddat, Luke W.
Schenk, Gerhard
Title Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry
Journal name Journal of Biological Inorganic Chemistry   Check publisher's open access policy
ISSN 0949-8257
1432-1327
Publication date 2014-03-01
Sub-type Article (original research)
DOI 10.1007/s00775-013-1079-0
Volume 19
Issue 3
Start page 389
End page 398
Total pages 10
Place of publication Heidelberg, Germany
Publisher Springer
Language eng
Formatted 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 (kcat, Km, and kcat/Km). 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.
Keyword Isothermal titration calorimetry
Kinetic assays
Metalloenzymes
Organophosphate-degrading enzymes
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 14 Feb 2014, 19:35:07 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences