Skip to main content
SpringerLink
Log in

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

  • Report
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Scheme 1

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

References

  1. Lipscomb JD (2008) Curr Opin Struct Biol 18:644–649

    Article  PubMed  CAS  Google Scholar 

  2. Vaillancourt FH, Bolin JT, Eltis LD (2006) Crit Rev Biochem Mol Biol 41:241–267

    Article  PubMed  CAS  Google Scholar 

  3. Bugg TD, Ramaswamy S (2008) Curr Opin Chem Biol 12:134–140

    Article  PubMed  CAS  Google Scholar 

  4. Koehntop KD, Emerson JP, Que L Jr (2005) J Biol Inorg Chem 10:87–93

    Article  PubMed  CAS  Google Scholar 

  5. Bruijnincx PCA, van Koten G, Klein Gebbink RJM (2008) Chem Soc Rev 37:2716–2744

    Article  PubMed  CAS  Google Scholar 

  6. Groce SL, Lipscomb JD (2005) Biochemistry 44:7175–7188

    Article  PubMed  CAS  Google Scholar 

  7. Groce SL, Miller-Rodeberg MA, Lipscomb JD (2004) Biochemistry 43:15141–15153

    Article  PubMed  CAS  Google Scholar 

  8. Vetting MW, Wackett LP, Que L Jr, Lipscomb JD, Ohlendorf DH (2004) J Bacteriol 186:1945–1958

    Article  PubMed  CAS  Google Scholar 

  9. Deeth RJ, Bugg TD (2003) J Biol Inorg Chem 8:409–418

    PubMed  CAS  Google Scholar 

  10. Kovaleva EG, Lipscomb JD (2007) Science 316:453–457

    Article  PubMed  CAS  Google Scholar 

  11. Siegbahn PEM, Haeffner F (2004) J Am Chem Soc 126:8919–8932

    Article  PubMed  CAS  Google Scholar 

  12. Reynolds MF, Costas M, Ito M, Jo D-H, Tipton AA, Whiting AK, Que L Jr (2003) J Biol Inorg Chem 8:263–272

    PubMed  CAS  Google Scholar 

  13. Borges F, Guimarães C, Lima JLFC, Pinto I, Reis S (2005) Talanta 66:670–673

    Article  PubMed  CAS  Google Scholar 

  14. Vaillancourt FH, Barbosa CJ, Spiro TG, Bolin JT, Blades MW, Turner RFB, Eltis LD (2002) J Am Chem Soc 124:2485–2496

    Article  PubMed  CAS  Google Scholar 

  15. Shu L, Chiou Y-M, Orville AM, Miller MA, Lipscomb JD, Que L Jr (1995) Biochemistry 34:6649–6659

    Article  PubMed  CAS  Google Scholar 

  16. Fielding AJ, Kovaleva EG, Farquhar ER, Lipscomb JD, Que L Jr (2011) J Biol Inorg Chem 16:341–355

    Article  PubMed  CAS  Google Scholar 

  17. Grossoehme NE, Spuches AM, Wilcox DE (2010) J Biol Inorg Chem 15:1183–1191

    Article  PubMed  CAS  Google Scholar 

  18. Leavitt S, Freire E (2001) Curr Opin Chem Biol 11:560–566

    CAS  Google Scholar 

  19. Goldberg RN, Kishore N, Lennen RM (2002) J Phys Chem Ref Data 31:231–370

    Article  CAS  Google Scholar 

  20. Emerson JP, Wagner ML, Reynolds MF, Que L Jr, Sadowsky MJ, Wackett LP (2005) J Biol Inorg Chem 10:751–760

    Article  PubMed  CAS  Google Scholar 

  21. Emerson JP, Kovaleva EG, Farquhar ER, Lipscomb JD, Que L Jr (2008) Proc Natl Acad Sci USA 105:7347–7352

    Article  PubMed  CAS  Google Scholar 

  22. Wang YZ, Lipscomb JD (1997) Protein Expr Purif 10:1–9

    Article  PubMed  CAS  Google Scholar 

  23. Miller MA, Lipscomb JD (1996) J Biol Chem 271:5524–5535

    Article  PubMed  CAS  Google Scholar 

  24. Wardman P (1989) J Phys Chem Ref Data 18:1637–1755

    Article  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Department of Chemistry, Mississippi State University, Mississippi State, MS, 39762, USA

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

Authors
  1. Kate L. Henderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vu H. Le
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edwin A. Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph P. Emerson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph P. Emerson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 215 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Henderson, K.L., Le, V.H., Lewis, E.A. et al. Exploring substrate binding in homoprotocatechuate 2,3-dioxygenase using isothermal titration calorimetry. J Biol Inorg Chem 17, 991–994 (2012). https://doi.org/10.1007/s00775-012-0929-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-012-0929-5

Keywords

Search

Navigation