The importance of threonine-301 from cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) in substrate binding as demonstrated by site-directed mutagenesis.
Threonine-301 from rabbit liver cytochromes P-450 (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydroxylase) has been replaced by histidine via site-directed mutagenesis. In the oxidized state the mutant P-450s exhibited typical low-spin type absorption spectra of P-450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral change was induced on addition of substrates for their wild-type counterparts. The mutant P-450s were also completely devoid of the hydroxylase activity. These findings suggest that threonine-301, which is highly conserved in P-450s and located at the distal heme surface, plays an important role in substrate binding.
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011817:Rabbits; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine
D013912:Threonine; 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