JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 7, pp 991–994

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

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

DOI: 10.1007/s00775-012-0929-5

Cite this article as:
Henderson, K.L., Le, V.H., Lewis, E.A. et al. J Biol Inorg Chem (2012) 17: 991. doi:10.1007/s00775-012-0929-5

Abstract

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.

Keywords

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

Abbreviations

ACES

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

CTD

Catechol 2,3-dioxygenase

DHBD

2,3-Dihydroxybiphenyl 1,2-dioxygenase

HEPES

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

HPCA

Homoprotocatechuate

HPCD

Homoprotocatechuate 2,3-dioxygenase

ITC

Isothermal titration calorimetry

MOPS

3-Morpholinopropane-1-sulfonic acid

4-NC

4-Nitrocatechol

PIPES

1,4-Piperazinediethanesulfonic acid

Supplementary material

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

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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