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
Report

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)

References

  1. 1.
    Lipscomb JD (2008) Curr Opin Struct Biol 18:644–649PubMedCrossRefGoogle Scholar
  2. 2.
    Vaillancourt FH, Bolin JT, Eltis LD (2006) Crit Rev Biochem Mol Biol 41:241–267PubMedCrossRefGoogle Scholar
  3. 3.
    Bugg TD, Ramaswamy S (2008) Curr Opin Chem Biol 12:134–140PubMedCrossRefGoogle Scholar
  4. 4.
    Koehntop KD, Emerson JP, Que L Jr (2005) J Biol Inorg Chem 10:87–93PubMedCrossRefGoogle Scholar
  5. 5.
    Bruijnincx PCA, van Koten G, Klein Gebbink RJM (2008) Chem Soc Rev 37:2716–2744PubMedCrossRefGoogle Scholar
  6. 6.
    Groce SL, Lipscomb JD (2005) Biochemistry 44:7175–7188PubMedCrossRefGoogle Scholar
  7. 7.
    Groce SL, Miller-Rodeberg MA, Lipscomb JD (2004) Biochemistry 43:15141–15153PubMedCrossRefGoogle Scholar
  8. 8.
    Vetting MW, Wackett LP, Que L Jr, Lipscomb JD, Ohlendorf DH (2004) J Bacteriol 186:1945–1958PubMedCrossRefGoogle Scholar
  9. 9.
    Deeth RJ, Bugg TD (2003) J Biol Inorg Chem 8:409–418PubMedGoogle Scholar
  10. 10.
    Kovaleva EG, Lipscomb JD (2007) Science 316:453–457PubMedCrossRefGoogle Scholar
  11. 11.
    Siegbahn PEM, Haeffner F (2004) J Am Chem Soc 126:8919–8932PubMedCrossRefGoogle Scholar
  12. 12.
    Reynolds MF, Costas M, Ito M, Jo D-H, Tipton AA, Whiting AK, Que L Jr (2003) J Biol Inorg Chem 8:263–272PubMedGoogle Scholar
  13. 13.
    Borges F, Guimarães C, Lima JLFC, Pinto I, Reis S (2005) Talanta 66:670–673PubMedCrossRefGoogle Scholar
  14. 14.
    Vaillancourt FH, Barbosa CJ, Spiro TG, Bolin JT, Blades MW, Turner RFB, Eltis LD (2002) J Am Chem Soc 124:2485–2496PubMedCrossRefGoogle Scholar
  15. 15.
    Shu L, Chiou Y-M, Orville AM, Miller MA, Lipscomb JD, Que L Jr (1995) Biochemistry 34:6649–6659PubMedCrossRefGoogle Scholar
  16. 16.
    Fielding AJ, Kovaleva EG, Farquhar ER, Lipscomb JD, Que L Jr (2011) J Biol Inorg Chem 16:341–355PubMedCrossRefGoogle Scholar
  17. 17.
    Grossoehme NE, Spuches AM, Wilcox DE (2010) J Biol Inorg Chem 15:1183–1191PubMedCrossRefGoogle Scholar
  18. 18.
    Leavitt S, Freire E (2001) Curr Opin Chem Biol 11:560–566Google Scholar
  19. 19.
    Goldberg RN, Kishore N, Lennen RM (2002) J Phys Chem Ref Data 31:231–370CrossRefGoogle Scholar
  20. 20.
    Emerson JP, Wagner ML, Reynolds MF, Que L Jr, Sadowsky MJ, Wackett LP (2005) J Biol Inorg Chem 10:751–760PubMedCrossRefGoogle Scholar
  21. 21.
    Emerson JP, Kovaleva EG, Farquhar ER, Lipscomb JD, Que L Jr (2008) Proc Natl Acad Sci USA 105:7347–7352PubMedCrossRefGoogle Scholar
  22. 22.
    Wang YZ, Lipscomb JD (1997) Protein Expr Purif 10:1–9PubMedCrossRefGoogle Scholar
  23. 23.
    Miller MA, Lipscomb JD (1996) J Biol Chem 271:5524–5535PubMedCrossRefGoogle Scholar
  24. 24.
    Wardman P (1989) J Phys Chem Ref Data 18:1637–1755CrossRefGoogle Scholar

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

Personalised recommendations