Amino acid residue 147 of human aldosterone synthase and 11beta-hydroxylase plays a key role in 11beta-hydroxylation.
A number of amino acids differ between aldosterone synthase and 11beta-hydroxylase. To assess their importance in determining the different functional specificities, we substituted aldosterone synthase-specific (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11beta-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493), respectively. I248T, Q43R, and T493M had no effect on steroid production compared to wild-type aldosterone synthase. However, CYP11B2-D147E caused a significant increase in corticosterone production and a smaller increase in aldosterone production from 11-deoxycorticosterone (DOC). This appeared to be predominantly due to an increase in the 11beta-hydroxylation of DOC to corticosterone mediated by a decrease in Km, which was 1.4 micromol/L for the mutant compared with 5 micromol/L for the wild-type enzyme. CYP11B2-D147E had no effect on the conversion of 11-deoxycortisol to cortisol. The reverse construct (CYP11B1-E147D), substituting the 11beta-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in Km that was 7.5 micromol/L for the mutant compared with 2.5 micromol/L for the wild-type enzyme. Again, the conversion of 11-deoxycortisol to cortisol was unimpaired. Thus, amino acid 147 is involved in the transformation of the 17-deoxysubstrate, but not the 17alpha-hydroxysubstrate. The results demonstrate that a conservative change in amino acid, even at some linear distance from known active centers, can significantly affect enzyme substrate affinity and subsequent steroid hormone production.
Fisher
A
A
Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, United Kingdom.
Fraser
R
R
Mc Connell
J
J
Davies
E
E
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics / Q000502:physiology*; D006900:Hydroxylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids / Q000235:genetics; D051381:Rats; D020303:Rats, Inbred Dahl; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D000596:Amino Acids; D015345:Oligonucleotide Probes; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase