Comparison of the hamster and human adrenal P450c17 (17 alpha-hydroxylase/17,20-lyase) using site-directed mutagenesis and molecular modeling.
In order to understand the activity specificity of the hamster cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17 alpha-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17 alpha-hydroxylation to 46% when incubated with progesterone.To comprehend the altered activity profiles of these hamster P450c17 mutants, we have elaborated a 3D model of the hamster P450c17 and compared it to our preceding model of the human P450c17. Analysis of the mutants with this model showed that, without direct contact to the substrates, these mutations transmit structural changes to the active site. By analogy, these results support the concept that any cellular changes modifying the external structure of P450c17, such as phosphorylation, could have influence on its active site and enzymatic activities.
Mathieu
Axel P
AP
Department of Biochemistry, Faculty of Medicine, University of, Sherbrooke, Que., Canada J1H 5N4.
Auchus
Richard J
RJ
LeHoux
Jean Guy
JG
D000311:Adrenal Glands / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase