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Characterization of the Catalytic Properties of Recombinant Acetohydroxyacid Synthase from Tobacco

  • Published : 2005.02.20

Abstract

The nature of the active site of Tobacco acetohydroxyacid synthase (AHAS) in the substrate- and cofactorbinding was studied by kinetics and fluorescence spectroscopy. The substrate saturation curve does not follow Michaelis-Menten kinetics at different temperatures (7, 21 and 37 ${^{\circ}C}$), pH (6.5, 7.5 and 8.5) and buffers (Tris-HCl and MOPS). The concentration of one half of the maximum velocity ($S_{0.5}$) decreased in the following order: pyruvate $\gt$ ThDP $\approx$$Mg^{+2}$ $\gt$ FAD. However, the catalytic efficiency (K$_{cat}/S_{0.5}$) inversely decreased in the following order; FAD $\gt$ $Mg^{+2}$ $\approx$ThDP $\gt$ pyruvate, indicating that the cofactors by in decreasing order; FAD, $Mg^{+2}$, ThDP, affect the catalysis of AHAS. The dissociation constant ($K_d$) of the intrinsic tryptophan fluorescence decreased with the same tendency of the concentration of one half of the maximum velocity ($S_{0.5}$) decreasing order. This data provides evidence that the substrate and cofactor binding natures of the active site, as well as its activation characteristics, resemble those of other ThDP-dependent enzymes.

Keywords

References

  1. Umbarger, H. Annu. Rev. Biochem. 1978, 47, 533-606 https://doi.org/10.1146/annurev.bi.47.070178.002533
  2. Duggleby, R.; Pang, S. J. Biochem. Mol. Biol. 2000, 33, 1-36
  3. Chang, Y.; Wang, A.; Cronan, J. J. Biol. Chem. 1993, 268, 3911- 3919
  4. Macheroux, P.; Schonbrunn, E.; Svergun, D.; Volkov, D.; Koch, M.; Bornemann, S.; Thomeley, R. Biochem. J. 1998, 335, 319- 327
  5. Pang, S.; Guddat, L.; Duggleby, R. Acta Crystallog. 2001, Sect. D 57, 1321-1323
  6. Pang, S.; Duggleby, R.; Guddat, L. J. Mol. Biol. 2002, 317, 249- 262 https://doi.org/10.1006/jmbi.2001.5419
  7. Yoon, M. Y.; Hwang, J. H.; Choi, M. K.; Back, D. K.; Kim, J.; Kim, Y. T.; Choi, J. D. FEBS Letters 2003, 555, 185-191 https://doi.org/10.1016/S0014-5793(03)01177-3
  8. Chong, C. K.; Shin, H. J.; Chang, S. I.; Choi, J. D. Biochem. Biophys. Res. Commun. 1999, 259, 136-140 https://doi.org/10.1006/bbrc.1999.0740
  9. Kim, J.; Baek, D. K.; Choi, J. D.; Kim, Y. T.; Yoon, M. Y. Biochem. J. 2004, 383, 1-10 https://doi.org/10.1042/BJ20040913
  10. Hwang, J.; Kim, J.; Kim, Y. T.; Choi, J. D.; Yoon, M. Y. Bull. Korean Chem. Soc. 2003, 24, 1856-1858 https://doi.org/10.5012/bkcs.2003.24.12.1856
  11. Chang, S. I.; Kang, M. K.; Choi, J. D.; Namgoong, S. K. Biochem. Biophys. Res. Commun. 1997, 234, 549-553 https://doi.org/10.1006/bbrc.1997.6678
  12. Westerfeld, W. W. J. Biol. Chem. 1943, 161, 495-502
  13. Lee, Y. T.; Chang, A. K.; Duggleby, R. G. FEBS Lett. 1999, 452, 341-345 https://doi.org/10.1016/S0014-5793(99)00668-7
  14. Dawson, R. M. C.; Elliott, D. C.; Elliott, N. H.; Johnes, K. M. Data for Biochemical Research, 2nd ed.; Oxford University: London, 1979; p 423
  15. Cleland, W. W. Methods Enzymol. 1979, 63, 103-147 https://doi.org/10.1016/0076-6879(79)63008-2
  16. Pang, S. S.; Duggleby, R. G.; Guddat, L. W. J. Mol. Biol. 2002, 317, 249-262 https://doi.org/10.1006/jmbi.2001.5419
  17. Schloss, J. V.; Van Dyk, D. E.; Vasta, J. F.; Kutny, R. M. Biochemistry 1985, 24, 4952-4959 https://doi.org/10.1021/bi00339a034
  18. Chang, A. K.; Duggleby, R. G. Biochem. J. 1998, 333, 767-777
  19. Miflin, B. J. Arch. Biochem. Biophys. 1971, 146, 542-550 https://doi.org/10.1016/0003-9861(71)90159-7
  20. Chang, A. K.; Duggleby, R. G. Biochem. J. 1997, 327, 161-169
  21. Yang, J. H.; Kim, S. S. Biochim. Biophys. Acta 1993, 1157, 178- 184 https://doi.org/10.1016/0304-4165(93)90062-D
  22. Choi, J. D.; Kim, B. H.; Yoon, M. Y. Bull. Korean Chem. Soc 2003, 24, 627-632 https://doi.org/10.5012/bkcs.2003.24.5.627

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