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.