Journal of Protein Chemistry

, Volume 12, Issue 1, pp 51–56 | Cite as

Diethylpyrocarbonate reactivity ofKlebsiella aerogenes urease: Effect ofpH and active site ligands on the rate of inactivation

  • Il-Seon Park
  • Robert P. Hausinger
Article

Abstract

Reaction ofKlebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. ThepH dependence for the rate of inactivation indicated that the reactive residue possessed apK a of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.

Key words

Urease diethylpyrocarbonate histidine reactivity active site modification 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andrews, R. F., Blakeley, R. L., and Zerner, B. (1988). InThe Bioinorganic Chemistry of Nickel (Lancaster, J. R., ed.), VCH Publishers, New York, pp. 141–165.Google Scholar
  2. Bloxham, D. P. (1981).Biochem. J. 193, 93–97.Google Scholar
  3. Carrillo, N., Arana, J. L., and Vallejos, R. H. (1981).J. Biol. Chem. 256, 6823–6828.Google Scholar
  4. Lee, M. H., Mulrooney, S. B., and Hausinger, R. P. (1990).J Bacteriol. 172, 4427–4431.Google Scholar
  5. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265–275.Google Scholar
  6. Lundblad, R. L., and Noyes, C. M. (1984).Chemical Reagents for Protein Modification, CRC Press, Boca Raton, Florida, pp. 101–125.Google Scholar
  7. Miles, E. W. (1977).Meth. Enzymol. 47, 431–442.Google Scholar
  8. Mobley, H. L. T., and Hausinger, R. P. (1989).Microbiol. Rev. 53, 85–108.Google Scholar
  9. Mülhrad, A., Hegyi, G., and Tóth, G. (1967).Acta Biochim. Biophys. Acad. Sci. Hung. 2, 19–29.Google Scholar
  10. Mulrooney, S. B., and Hausinger, R. P. (1990).J. Bacteriol. 172, 5837–5843.Google Scholar
  11. Mulrooney, S. B., Pankratz, H. S., and Hausinger, R. P. (1989).J. Gen. Microbiol. 135, 1769–1776.Google Scholar
  12. Todd, M. J., and Hausinger, R. P. (1987).J. Biol. Chem. 262, 5963–5967.Google Scholar
  13. Todd, M. J., and Hausinger, R. P. (1989).J. Biol. Chem. 264, 15,835–15,842.Google Scholar
  14. Todd, M. J., and Hausinger, R. P. (1991a).J. Biol. Chem. 266, 10,260–10,267.Google Scholar
  15. Todd, M. J., and Hausinger, R. P. (1991b).J. Biol. Chem. 266, 24,327–24,331.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Il-Seon Park
    • 1
  • Robert P. Hausinger
    • 1
  1. 1.Departments of Microbiology and BiochemistryMichigan State UniversityEast Lansing

Personalised recommendations