AFFINITY LABELING IDENTIFIES HISTIDINE AT THE ACTIVE-SITE OF HUMAN PLACENTAL 3-BETA-HYDROXYSTEROID DEHYDROGENASE AND STEROID 5-]4-ENE-ISOMERASE

OBJECTIVE: Our aim was to determine if the multifunction enzyme, 3beta -hydroxysteroid dehydrogenase and steroid 5 --> 4-ene-isomerase has on e or more active sites to effect dehydrogenase and isomerase activitie s. STUDY DESIGN: This steroid, which we have purified to homogeneity f rom human placental microsomes, was inactivated by the affinity labeli ng steroid, 2alpha-bromo[2'-C-14]acetoxyprogesterone. The amino acids that were radioalkylated in the absence and presence of the dehydrogen ase substrate pregnenolone were identified. RESULTS: Pregnenolone comp letely abolished the inactivation of dehydrogenase. Histidine was loca lized in the active site of 3beta-hydroxysteroid dehydrogenase because the radiolabel disappeared from enzyme inactivated in the presence of pregnenolone. Cysteine, a major radiolabeled product (80%) in the abs ence of pregnenolone, was decreased twofold in incubations that contai ned pregnenolone. Neither pregnenolone nor the isomerase substrate 5-a ndrostene-3,17-dione protected isomerase from inactivation by the affi nity alkylator. CONCLUSION: This observation contradicts coexisting, s eparate binding sites, one for each activity. Rather, a conformation s hift around one binding region prompted by products of the dehydrogena se reaction may create the isomerase activity.