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Amino Acid Residue 147 of Human Aldosterone Synthase and 11ß-Hydroxylase Plays a Key Role in 11ß-Hydroxylation¹

Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, United Kingdom G11 6NT

Address all correspondence and requests for reprints to: Dr. R. Fraser, Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, United Kingdom G11 6NT. E-mail: rfraser{at}clinmed.gla.ac.uk

A number of amino acids differ between aldosterone synthase and 11ß-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 11ß-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 11ß-hydroxylation of DOC to corticosterone mediated by a decrease in K_m, which was 1.4 µmol/L for the mutant compared with 5 µmol/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 11ß-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in K_m that was 7.5 µmol/L for the mutant compared with 2.5 µmol/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 17-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.

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