Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human 5-lipoxygenase.
Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.
Ishii
S
S
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa.
Noguchi
M
M
Miyano
M
M
Matsumoto
T
T
Noma
M
M
D000595:Amino Acid Sequence; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019159:Nonheme Iron Proteins; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007501:Iron; D008084:Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase