The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4

Arch Biochem Biophys. 2000 Feb 15;374(2):269-78. doi: 10.1006/abbi.1999.1599.


The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Base Sequence
  • Binding Sites
  • Catalytic Domain
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • DNA Primers
  • DNA, Complementary
  • Humans
  • Ketosteroids / metabolism
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Progesterone / metabolism
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Testosterone / metabolism


  • DNA Primers
  • DNA, Complementary
  • Ketosteroids
  • Recombinant Proteins
  • Testosterone
  • Progesterone
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human