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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 33, Number 9
BKCSDE 33(9)
September 20, 2012 


Rapid Mapping of Active Site of KSI by Paramagnetic NMR

Yong Nam Joe, Hyung Jin Cha, Hyeong Ju Lee, Kwan Yong Choi, Hee Cheon Lee*
NMR, Ketosteroid isomerase, Active site, Paramagnetic agent, Equilenin
Active site mapping has been done for Δ5-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on 1H-15N 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.
2981 - 2984
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