Biochemical Characteristics of a Palmitoyl Acyl Carrier Protein Thioesterase Purified from Iris pseudoacorus

  • Kang, Han-Chul (Division of Biochemistry, National Agricultural Science and Technology Institute, Rural Development Administration) ;
  • Hwang, Young-Soo (Division of Biochemistry, National Agricultural Science and Technology Institute, Rural Development Administration)
  • Received : 1996.05.15
  • Published : 1996.09.30

Abstract

The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

Keywords

active site;co-gradient protein elution;palmitoyl acyl carrier protein thioesterase

References

  1. Plant Mol. Biol. v.20 Amanda, H.;Peter, F.L.;Antoni, R.S. https://doi.org/10.1007/BF00027148
  2. Method Enzymol. v.35 Barnes, E.M.
  3. Anal. Biochem. v.72 Bradford, M.M. https://doi.org/10.1016/0003-2697(76)90527-3
  4. Arch. Biochem. Biophys. v.290 Davies, H.M.;Anderson, L.;Fan, C.;Hawkins, D.J. https://doi.org/10.1016/0003-9861(91)90588-A
  5. Arch. Biochem. Biophys. v.205 De Renobales, M.;Rogers, L.;Kolattukudy, P. https://doi.org/10.1016/0003-9861(80)90129-0
  6. Planta v.189 Dormann, P.;Spencer, F.;Ohlrogge, J.B. https://doi.org/10.1007/BF00194441
  7. Nature v.227 Laemmli, U.K. https://doi.org/10.1038/227680a0
  8. J. Biol. Chem. v.253 Lin, C.Y.;Smith, S.
  9. J. Biol. Chem. v.257 Mckeon, T.A.;Stumpf, P.K.
  10. Arch. Biochem. Biophys. v.189 Ohlrogge, J.B.;Shine, W.E.;Stumpf, P.K. https://doi.org/10.1016/0003-9861(78)90225-4
  11. Arch. Biochem. Biophys. v.284 Pollard, M.R.;Anderson, L.;Fan, C.Hawkins, D.J.;Davies, H.M. https://doi.org/10.1016/0003-9861(91)90300-8
  12. Eur. J. Biochem. v.162 Randhawa, Z.I.;Naggert, J.;Blacher, R.W.;Smith, S. https://doi.org/10.1111/j.1432-1033.1987.tb10678.x
  13. J. Biol. Chem. v.254 Rock, C.O.;Garwin, J.L.
  14. J. Biol. Chem. v.257 Rogers, L.;Kolattukudy, P.E.;de Renobales, M.
  15. Biochemistry v.25 Seay, T.;Lueking, D.R. https://doi.org/10.1021/bi00357a029
  16. Biochem. Soc. Trans. v.14 Smith, S.;Mikkelsen, J.;Witkowski, A.;Libertini, L.J. https://doi.org/10.1042/bst0140583
  17. Science v.257 Voelker, T.A.;Norrell, A.C.;Anderson, L.;Bleibaum, J.;Fan, C.;Hankins, D.J.;Radke, S.E.;Davies, H.M. https://doi.org/10.1126/science.1621095