A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase

Biophys J. 2009 Jun 17;96(12):5003-12. doi: 10.1016/j.bpj.2009.04.014.


Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k(cat)/K(M,IPM)) for the substrate IPM has an unusual temperature dependence, showing a local minimum at approximately 35 degrees C. In search of an explanation, we measured the individual constants k(cat) and K(M,IPM) as a function of temperature, and found that the van 't Hoff plot of K(M,IPM) shows sigmoid-like transition in the 20-40 degrees C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30 degrees C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Isopropylmalate Dehydrogenase / chemistry*
  • 3-Isopropylmalate Dehydrogenase / metabolism
  • Biocatalysis*
  • Calorimetry
  • Calorimetry, Differential Scanning
  • Circular Dichroism
  • Deuterium Exchange Measurement
  • Escherichia coli / enzymology*
  • Fluorescence Resonance Energy Transfer
  • Malates / chemistry
  • Malates / metabolism
  • Models, Molecular
  • NAD / chemistry
  • NAD / metabolism
  • Protein Binding
  • Protein Structure, Quaternary
  • Substrate Specificity
  • Temperature*


  • Malates
  • NAD
  • beta-isopropylmalate
  • 3-Isopropylmalate Dehydrogenase