Purification, characterization and catalytic properties of human sterol 8-isomerase

W. David Nes, Wenxu Zhou, Allen L. Dennis, Haoxia Li, Zhonghua Jia, Richard A. Keith, Timothy M. Piser, Stephen T. Furlong

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

CHO2, encoding human sterol 8-isomerase (hSI), 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 Km of 50 μM 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 cholest8(14)-enol, whereas the enzyme-generated product was a Δ7sterol, suggesting absolute stereochemical control of the reaction by hSI. Using 2H2O 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-9α was established by a combination of 1H- and 13C-NMR analyses of the enzyme-generated cholesta7,24-dienol. Kinetic analyses indicated the reaction equilibrium (Keq = 14; ΔGo′ = - 6.5 kJ/mol) for double-bond isomerization favoured the forward direction, δ8 to Δ7. Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants (Ki): emopamil (2 μM) = tamoxifen (1 μM) = tridemorph (1 μM) < 25-azacholesterol (21 μM) < ketoconazole (156 μM) < cholesterol (620 μM). The results were consistent with stereoelectronic features of isomerization and support the general model for Δ7-sterol formation in cholesterol synthesis.

Original languageEnglish
Pages (from-to)587-599
Number of pages13
JournalBiochemical Journal
Volume367
Issue number3
DOIs
StatePublished - Nov 1 2002

Keywords

  • Cholesterol
  • Emopamil
  • Isomerization reaction
  • Lanosterol
  • Stereochemistry

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