期刊
BIOCHEMICAL JOURNAL
卷 367, 期 -, 页码 587-599出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20020551
关键词
cholesterol; emopamil; isomerization reaction; lanosterol; stereochemistry
资金
- NIGMS NIH HHS [GM63477] Funding Source: Medline
CHO2, encoding human sterol 8-isomerase (hSl), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K-m of 50 muM 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 (H2O)-H-2 and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GG-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of H-1- and C-13-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 hSl with different high-energy intermediate analogues produced the following dissociation constants (K-i): emopamil (2 muM) = tamoxifen (1 muM) = tridemorph (1 muM) < 25-azacholesterol (21 muM) < ketoconazole (156 muM) < cholesterol (620 muM). The results were consistent with stereoelectronic features of isomerization and support the general model for Delta(7)-sterol formation in cholesterol synthesis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据