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J Clin Endocrinol Metab ; 85(3): 1261-6, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10720073

ABSTRACT

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.


Subject(s)
Amino Acids/physiology , Cytochrome P-450 CYP11B2/physiology , Steroid 11-beta-Hydroxylase/physiology , Animals , COS Cells , Chlorocebus aethiops , Cytochrome P-450 CYP11B2/genetics , Hydroxylation , Kinetics , Mutagenesis, Site-Directed , Oligonucleotide Probes , Plasmids/genetics , Rats , Rats, Inbred Dahl , Steroid 11-beta-Hydroxylase/genetics , Transfection
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