Abstract
Attempts to explore the structural basis for functions of cytochrome P-450 at the DNA level were outlined briefly and, as an example of such approaches, our studies on laurate (ω-1)-hydroxylase (P-450(ω-1)) were described. P-450(ω-1) exhibits 81 % similarity in the primary structure to testosterone 16 α-hydroxylase. Chimeras of the both P-450s were synthesized in yeast cells transformed with plasmids constructed for expression of chimeric P-450 cDNAs and their spectral and catalytic properties were examined. The region spanning about 50 residues is essential to the binding of substrates (laurate and caprate) for P-450(ω-1) was found. In addition to the sequence enough to bind the substrates, the segment of about 35 residues is necessery for the hydroxylase activity. Threonine-301 of P-450(ω-1), which is highly conserved in all P-450s and located at the distal heme surface trans to the thiolate ligand, was substituted by His, Val, Ser or Ala via site-directed mutagenesis. The addition of the fatty acids to ferric P-450(ω-1) induced spectral change ascribable to the bindig of substrates in the Val-or Ser-mutant as well as the wild-type P-450 but did not in the His-or Ala-mutant. These mutants were also devoid of the hydroxylase activity. On the other hand, substrate specificity of P-450(ω-1) was altered by substitution of Val or Ser for Thr-301. These findings indicate that residues (or atoms) at the γ-position of the amino acid-301 is important to the substrate interaction.
