Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Rapid Mapping of Active Site of KSI by Paramagnetic NMR

  • Joe, Yong-Nam (Department of Chemistry, Pohang University of Science and Technology) ;
  • Cha, Hyung-Jin (Department of Life Science, Pohang University of Science and Technology) ;
  • Lee, Hyeong-Ju (Department of Chemistry, Pohang University of Science and Technology) ;
  • Choi, Kwan-Yong (Department of Life Science, Pohang University of Science and Technology) ;
  • Lee, Hee-Cheon (Department of Chemistry, Pohang University of Science and Technology)
  • Received : 2012.06.02
  • Accepted : 2012.06.12
  • Published : 2012.09.20
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Abstract

Active site mapping has been done for ${\Delta}^5$-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on $^1H-^{15}N$ HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similar to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI, and can be applied to the analysis of both active site and function in unknown protein.

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References

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