Identification of regions functioning in substrate interaction of rabbit liver cytochrome P-450 (laurate (omega-1)-hydroxylase).
The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (omega-1) hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16 alpha-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low-and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding of the fatty acids to the substrate site of the chimeric P-450s. However the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (omega-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450.(ABSTRACT TRUNCATED AT 250 WORDS)
Uno
T
T
Institute for Protein Research, Osaka University.
Imai
Y
Y
D000818:Animals; D001483:Base Sequence; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D008099:Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011817:Rabbits; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D039181:Steroid 16-alpha-Hydroxylase; D013347:Subcellular Fractions / Q000201:enzymology
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D039181:Steroid 16-alpha-Hydroxylase; D042926:Cytochrome P-450 CYP4A