| 12133002 |
Nes WD, Zhou W, Dennis AL, Li H, Jia Z, Keith RA, Piser TM, Furlong ST: Purification, characterization and catalytic properties of human sterol 8-isomerase. Biochem J. 2002 Nov 1;367(Pt 3):587-99.
CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s (-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8 (14)-enol, whereas the enzyme-generated product was a Delta (7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2) H (2) O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1) H- and (13) C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG (o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta (8) to Delta (7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM) (156 microM)cholesterol synthesis. |
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