The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4
| Autor: | Fabienne Roussel, Kishore K. Khan, James R. Halpert |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular DNA Complementary Stereochemistry Protein Conformation Metabolite medicine.medical_treatment Molecular Sequence Data Biophysics Biochemistry Steroid Mixed Function Oxygenases Substrate Specificity Hydroxylation chemistry.chemical_compound Cytochrome P-450 Enzyme System Catalytic Domain Cytochrome P-450 CYP3A medicine Humans Testosterone Amino Acid Sequence Binding site Site-directed mutagenesis Molecular Biology Progesterone DNA Primers Binding Sites biology Base Sequence Active site Cytochrome P450 Ketosteroids Recombinant Proteins chemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed |
| Zdroj: | Archives of biochemistry and biophysics. 374(2) |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect