JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 7, pp 991–994 | Cite as

Exploring substrate binding in homoprotocatechuate 2,3-dioxygenase using isothermal titration calorimetry

  • Kate L. Henderson
  • Vu H. Le
  • Edwin A. Lewis
  • Joseph P. Emerson


Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K = 1.1 ± 0.6 × 106) and enthalpy change (ΔH = −17.0 ± 1.7 kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.


Isothermal titration calorimetry Extradiol dioxygenase Substrate binding Two histidine/one carboxylate facial triad Non-heme iron 



N-(2-acetamido)-2-aminoethanesulfonic acid


Catechol 2,3-dioxygenase


2,3-Dihydroxybiphenyl 1,2-dioxygenase


4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid




Homoprotocatechuate 2,3-dioxygenase


Isothermal titration calorimetry


3-Morpholinopropane-1-sulfonic acid




1,4-Piperazinediethanesulfonic acid



The authors thank Whitnee Simmons for helping to initiate this project and John Lipscomb for the B. fuscum HPCD expression system and helpful discussions regarding our ITC data.

Supplementary material

775_2012_929_MOESM1_ESM.pdf (215 kb)
Supplementary material 1 (PDF 215 kb)


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Copyright information

© SBIC 2012

Authors and Affiliations

  • Kate L. Henderson
    • 1
  • Vu H. Le
    • 1
  • Edwin A. Lewis
    • 1
  • Joseph P. Emerson
    • 1
  1. 1.Department of ChemistryMississippi State UniversityMississippi StateUSA

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