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Affinity alkylators, 11. cap alpha. -bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common active site-histidine in human placental 17. beta. ,20-. cap alpha. -hydroxysteroid dehydrogenase

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5509159
; ;

Purified human placental 17..beta..,20..cap alpha..-hydroxysteroid dehydrogenase (17,20-HSD), after complete inactivation by estrone 3-bromoacetate (3-BAE) in the presence of NADPH, was reactivated to 100% activity by base-catalyzed hydrolysis of the steroidal ester-enzyme conjugate and then repurified. Computer modeling predicted that 3-BAE and 11..cap alpha..-bromoacetoxyprogesterone (11-BAP) alkylate a common region of the enzyme active site. Kinetic studies argued that reactivated enzyme (RE) and native enzyme (NE) bind 11-BAP in the same orientation. 11-/sup 14/C-BAP produced 5-fold less radiolabeled 3-(carboxymethyl)histidine (3-CM-His) in RE than in NE. Despite having the same affinity for RE and NE, 11-BAP re-inactivated RE5-fold slower than NE. These results demonstrate that the nonradiolabeled 3-CM-His originally produced by 3-BAE in the enzyme active site hindered radioalkylation of this histidyl reside in RE by 11-/sup 14/C-BAP. Thus, 11-BAP and 3-BAE modify a common histidine in the enzyme active site, and this is direct evidence that the estradiol 17..beta..-dehydrogenase and 20..cap alpha..-hydroxysteroid dehydrogenase activities of 17,20-HSD reside at a single locus on one protein.

Research Organization:
Washington Univ. School of Medicine, St. Louis, MO
OSTI ID:
5509159
Report Number(s):
CONF-8604222-
Journal Information:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:3; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKYLATION
AMINO ACIDS
ANIMALS
AZOLES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
COENZYMES
COMPUTERIZED SIMULATION
ENZYME ACTIVITY
ENZYMES
ESTRANES
ESTROGENS
ESTRONE
FETAL MEMBRANES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
HORMONES
HYDROXY COMPOUNDS
IMIDAZOLES
ISOTOPE APPLICATIONS
KETONES
KINETICS
LABELLED COMPOUNDS
MAMMALS
MAN
MEMBRANES
NADP
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PLACENTA
PREGNANES
PRIMATES
PROGESTERONE
PURIFICATION
REACTION KINETICS
SIMULATION
STEROID HORMONES
STEROIDS
TRACER TECHNIQUES
VERTEBRATES