Point mutations at threonine-301 modify substrate specificity of rabbit liver microsomal cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase).
Thr-301 of cytochrome P-450 (laurate (omega-1)-hydroxylase) was replaced by Ser, Val, Ile, or Asn via site-directed mutagenesis. The Ser-, Val-, and Asn-mutants had lower laurate (omega-1)-hydroxylase activities than the wild-type P-450. The mutation to Ser did not affect caprate (omega-1)-hydroxylase activity and rather increased caprate omega-hydroxylase activity, but the Val- and Asn-mutants could not hydroxylate caprate. The Ile-mutant was devoid of the hydroxylase activities. The mutation also led to changes in the affinities for the fatty acids and exogenous ligands. Replacement of Thr-301 of cytochrome P-450 (testosterone 16 alpha-hydroxylase) by Ser or Val also affected the activities toward testosterone and progesterone in different ways. These findings indicate that residue 301 of the P-450s plays an important role in determining their substrate specificities.
Imai
Y
Y
Institute for Protein Research, Osaka University, Japan.
Nakamura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001216:Asparagine; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D003652:Decanoic Acids / Q000378:metabolism; D007532:Isoleucine; D007850:Lauric Acids / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011374:Progesterone / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine; D013053:Spectrophotometry; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D013912:Threonine; D014170:Transformation, Genetic; D014633:Valine
D003652:Decanoic Acids; D007850:Lauric Acids; D007532:Isoleucine; C030358:lauric acid; D013912:Threonine; D013739:Testosterone; D012694:Serine; D011374:Progesterone; D001216:Asparagine; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster; D042926:Cytochrome P-450 CYP4A; D014633:Valine