Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
권호 표지
DOI QR코드

DOI QR Code

Site-directed Mutagenesis of the Evolutionarily Conserved Tyr8 Residue in Rice Phi-class Glutathione S-transferase F3

  • Jo, Hyun-Joo (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Sciences, Chung-Ang University) ;
  • Pack, Mi-Jin (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Sciences, Chung-Ang University) ;
  • Seo, Jin-Young (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Sciences, Chung-Ang University) ;
  • Lim, Jin-Kyung (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Sciences, Chung-Ang University) ;
  • Kong, Kwang-Hoon (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Sciences, Chung-Ang University)
  • Received : 2013.05.27
  • Accepted : 2013.06.17
  • Published : 2013.09.20
Download PDF
⟨ Previous Next ⟩

Abstract

To elucidate the role of the evolutionarily conserved Tyr8 residue in rice Phi-class GSTF3, this amino acid was replaced with alanine and phenylalanine by site-directed mutagenesis, respectively. The replacement of Tyr8 with Ala significantly affected the catalytic activity and the kinetic parameters, whereas the substitutions of Tyr8 with Phe had almost no effect. The Y8A mutant resulted in approximately 90-100% decrease of the specific activity. Moreover, the Y8A mutant resulted approximately in 2-fold increase of $K_m$, approximately 60-80% decrease of $k_{cat}$, and approximately 6.5-fold decrease in $k_{cat}/K_m$. From the pH/log $k_{cat}/K_m$ plot, $pK_a$ values of the GSH in the wild-type enzyme-GSH complex, Y8A-GSH complex and Y8F-GSH complex were estimated to be approximately 6.8, 8.5 and 6.9, respectively. From these results, we suggest that the evolutionarily conserved Tyr8 residue in OsGSTF3 seems to influence the structural stability of the active site of OsGSTF3 rather than directly its catalytic activity.

Keywords

References

  1. Mannervik, B. Adv. Enzymol. Rel. Areas Mol. Biol. 1985, 57, 357.
  2. Mannervik, B.; Danielson, U. H. CRC Crit. Rev. Biochem. 1988, 23, 283. https://doi.org/10.3109/10409238809088226
  3. Fahey, R. C.; Sundquist, A. R. Adv. Enzymol. Rel. Areas Mol. Biol. 1991, 64, 1.
  4. Dixon, D. P.; Lapthorne, A.; Edwards, R. Genome Biol. 2002, 3, 3004.1.
  5. Dixon, D. P.; Cole, D. J.; Edwards, R. Plant Mol. Biol. 1998, 36, 75. https://doi.org/10.1023/A:1005958711207
  6. Dixon, D. P.; Davies, B. G.; Edwards, R. J. Biol. Chem. 2002, 277, 30859. https://doi.org/10.1074/jbc.M202919200
  7. Edwards, R.; Dixon, D. P.; Walbot, V. Trends Plant Sci. 2000, 5,193. https://doi.org/10.1016/S1360-1385(00)01601-0
  8. Droog, F. J. Palt Growth Regul. 1997, 16, 95. https://doi.org/10.1007/PL00006984
  9. Board, P. G.; Menon, D. Biochim. Biophys. Acta 2013, 1830, 3267. https://doi.org/10.1016/j.bbagen.2012.11.019
  10. Cho, H.-Y.; Kong, K.-H. Pestic. Biochem. Phys. 2005, 83, 29. https://doi.org/10.1016/j.pestbp.2005.03.005
  11. Yoon, S.-Y.; Kong, J.-N.; Jo, D.-H.; Kong, K.-H. Food Chem. 2011, 129, 1327. https://doi.org/10.1016/j.foodchem.2011.06.054
  12. Jo, H.-J.; Lee, J.-W.; Noh, J.-S.; Kong, K.-H. Bull. Korean Chem. Soc. 2012, 33, 4169. https://doi.org/10.5012/bkcs.2012.33.12.4169
  13. Habig, W. H.; Jakoby, W. B. Methods Enzymol. 1981, 77, 398. https://doi.org/10.1016/S0076-6879(81)77053-8
  14. Flohe, L.; Gunzler, W. A. Methods Enzymol. 1984, 105, 114. https://doi.org/10.1016/S0076-6879(84)05015-1
  15. Kong, K.-H.; Takasu, K.; Inoue, H.; Takahashi, K. Biochem. Biophys. Res. Commun. 1992, 184, 194. https://doi.org/10.1016/0006-291X(92)91177-R
  16. Kong, K.-H.; Nishida, M.; Inoue, H.; Takahashi, K. Biochem. Biophys. Res. Commun. 1992, 182, 1122. https://doi.org/10.1016/0006-291X(92)91848-K
  17. Barycki, J. J.; Colman, R. F. Biochemistry 1993, 32, 13002. https://doi.org/10.1021/bi00211a008
  18. Cummins, I.; Dixon, D. P.; Freitag-Pohl, S.; Skipsey, M.; Edwards, R. Drug Metab. Rev. 2011, 43, 266. https://doi.org/10.3109/03602532.2011.552910

Cited by

  1. Characterization of Site-specific Mutations in Human Dihydrolipoamide Dehydrogenase Significantly Destabilizing the Transition State of the Enzyme Catalysis vol.59, pp.4, 2015, https://doi.org/10.5012/jkcs.2015.59.4.344
  2. Characterization of a Naturally Occurring Mutation (Arg-447 to Gly) in Human Dihydrolipoamide Dehydrogenase of Patients with Neurological Deterioration vol.60, pp.1, 2016, https://doi.org/10.5012/jkcs.2016.60.1.88
  3. Characterization of Site-Specific Mutations Affecting the Catalytic Efficiency of Human Dihydrolipoamide Dehydrogenase toward NAD+ vol.59, pp.2, 2013, https://doi.org/10.5012/jkcs.2015.59.2.183