BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지

Chemical Modification of Residue of Lysine, Tryptophan, and Cysteine in Spinach Glycolate Oxidase

  • Lee, Duk-Gun (Department of Biochemistry, Chungbuk National University) ;
  • Cho, Nam-Jeong (Department of Biochemistry, Chungbuk National University) ;
  • Choi, Jung-Do (Department of Biochemistry, Chungbuk National University)
  • Received : 1996.03.04
  • Published : 1996.07.31
Download PDF
⟨ Previous Next ⟩

Abstract

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

Keywords

References

  1. Biochemistry v.5 Anderson, B.M.;Anderson, L.D.;Churchich, J.E. https://doi.org/10.1021/bi00873a017
  2. Anal. Biochem. v.12 Bradford, M.M.
  3. Chae, D.Y.
  4. Korean Biochem. J. (presently J. Biochem. Mol. Biol.) v.23 Choi, J.D.;Park, K.S.;Yang, D.J.
  5. Adv. Enzymol. v.45 Clelend, W.W.
  6. The Enzymes, Vol. 1 Cohen, I.A.;Boyer, P.D.(ed.)
  7. Chemical Modification of Enzymes: Active Site Studies Eyzaquirce, J.;Eyzaquirre, J.(ed.)
  8. J. Biol. Chem. v.241 Frigerio, N.A.;Harbury, H.A.
  9. Phytochemistry v.24 Hall, N.P.;Reggiani, R.;Lea, P.J. https://doi.org/10.1016/S0031-9422(00)82527-3
  10. Phytochemistry v.14 Kerr, M.W.;Groves, D.
  11. Korean Biochem. J. (presently J. Biochem. Mol. Biol.) v.19 Lee, K.K.;Choi, J.D.
  12. Bull. Korean Chem. Soc. v.8 Lee, K.K.;Kim, H.S.;Choi, J.D.
  13. Proc. Natl. Acad. Sci. USA v.82 Lindqvist, Y.;Branden, C.I. https://doi.org/10.1073/pnas.82.20.6855
  14. J. Biol. Chem. v.264 Lindqvist, Y.;Branden, C.I.
  15. Chemical Reagents for Protein Modification, Vol. 1 Lundblad, R.L.;Noyes, C.M.
  16. Annu. Rev. Biochem. v.52 Miziorko, H.M.;Lozimer, G.H. https://doi.org/10.1146/annurev.bi.52.070183.002451
  17. J. Biol. Chem. v.255 Ozawa, H.;Fujioka, M.
  18. Arch. Biochem. Biophys. v.154 Riao, L.L.;Richardson, K.E.
  19. Biochim. Biophys. Acta v.227 Schuman, M.;Massey, V. https://doi.org/10.1016/0005-2744(71)90003-9
  20. Korean Biochem. J. (presently J. Biochem. Mol. Biol.) v.27 Son, E.D.;Park, Y.S.;Cho, T.J.;Choi, J.D.;Cho, N.J.
  21. J. Biol. Chem. v.262 Volokita, M.;Somervile, C.B.
  22. Plant Physiol. v.41 Zelitch, I. https://doi.org/10.1104/pp.41.10.1623