Rc. Strickler et al., AFFINITY LABELING IDENTIFIES HISTIDINE AT THE ACTIVE-SITE OF HUMAN PLACENTAL 3-BETA-HYDROXYSTEROID DEHYDROGENASE AND STEROID 5-]4-ENE-ISOMERASE, American journal of obstetrics and gynecology, 168(4), 1993, pp. 1216-1222
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.