Histidine(77), glutamic acid(81), glutamic acid(123), threonine(126), asparagine(194), and tryptophan(197) of the human emopamil binding protein are required for in vivo sterol Delta 8-Delta 7 isomerization

The human emopamil binding protein (hEBP) exhibits sterol Delta 8-Delta 7 i somerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Del ta 8-Delta 7 isomerization-deficient erg2-3 yeast strain. Ala scanning muta genesis was used to identify residues in the four putative transmembrane al pha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta 5,7-ster ols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A -, and W197A-expressing yeast strains contained 10% or less of wild-type (w t) Delta 5,7-sterols. All substitutions of these six residues with function ally or structurally similar amino acid residues failed to fully restore ca talytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E 123D, still bound the enzyme inhibitor H-3-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His(77), Glu(81), Glu(123), Thr(126), Asn(194), and Trp(197) are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are propos ed to line the catalytic cleft. Ala mutants of Trp(102), Tyr(105), Asp(109) , Arg(111), and Tyr(112) in a conserved cytoplasmic domain (WKEYXKGDSRY) be tween transmembrane segments 2 and 3 contained less than 10% of wt Delta 5, 7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional co nsequences of mutations in sterol modifying enzymes.