Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis.
Cholesterol 7alpha-hydroxylase (cholesterol-NADPH oxidoreductase, EC 1.14.13.17, 7alpha-hydroxylating) is known to have extremely sensitive sulfhydryl group(s). It is believed that a cysteine residue that has a sulfhydryl group plays an important role in the decrease of this enzyme activity. The amino acid sequences of cholesterol 7alpha-hydroxylase of five different mammalian species, human, rat, rabbit, hamster and mouse, revealed that these mammalian species contain eight cysteine residues that are well conserved. To identify which cysteine residues are responsible for the extremely high lability, we used the technique of the site-directed mutagenesis. Eight mutated genes of human cholesterol 7alpha-hydroxylase in which one codon for a cysteine residue was changed to that for alanine were prepared and expressed in COS-1 cells. The protein mass and enzyme activity of cholesterol 7alpha-hydroxylse obtained from these eight mutated genes were determined. While all mutated genes expressed the enzyme mass, two mutated genes did not express protein capable of catalyzing 7alpha-hydroxylation of cholesterol: in one mutant a codon for the 7th cysteine residue (Cys 444) was substituted to that for alanine and in the other mutant a codon for the 8th cysteine residue (Cys 476) was changed similarly. These results suggest that the 7th and 8th cysteine residues are important for expression of the enzyme activity. Based on the fact that Cys 444 exists in the heme binding region, Cys 476 was suggested to be responsible for enzyme lability.
Tanaka
Shun-Ichi
S
Department of Surgery I, Miyazaki Medical College, 5200 Kiyotake, 889-1692, Miyazaki, Japan.
Kinowaki
Mitsuru
M
Eto
Tada-Aki
TA
Ota
Yoshikazu
Y
Maeda
Yorio
Y
Okuda
Kyu-Ichiro
K
Setoguchi
Toshiaki
T
Chijiiwa
Kazuo
K
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002790:Cholesterol 7-alpha-Hydroxylase; D003545:Cysteine