Reactivation of human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase: affirmation of affinity labeling principles.
Human placental 17 beta, 20 alpha-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 repurified 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, 16 alpha-bromoacetoxyestradiol-17 beta 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 17 beta, 20 alpha-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.
D015089:20-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000345:Affinity Labels / Q000494:pharmacology*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D006894:Hydroxyestrones / Q000138:chemical synthesis / Q000494:pharmacology; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding
D000345:Affinity Labels; D006894:Hydroxyestrones; C034312:estrone 3-bromoacetate; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase