Reactivation of human placental 17β,20α-hydroxysteroid dehydrogenase: Affirmation of affinity labeling principles

Article abstract of DOI:10.1016/0039-128X(84)90039-4

Human placental 17β, 20α-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was purified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16α-bromacetoxyestradiol-17β 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17β, 20α-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.