Purification, characterization and catalytic properties of human sterol 8-isomerase.
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) <ketoconazole (156 microM)<cholesterol (620 microM). The results were consistent with stereoelectronic features of isomerization and support the general model for Delta(7)-sterol formation in cholesterol synthesis.
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA. wdavid.nes@ttu.edu
W David
WD
Nes
Wenxu
W
Zhou
Allen L
AL
Dennis
Haoxia
H
Li
Zhonghua
Z
Jia
Richard A
RA
Keith
Timothy M
TM
Piser
Stephen T
ST
Furlong
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013251:Steroid Isomerases; C022669:delta(8)-delta(7)-sterol isomerase