Human placental estradiol 17. beta. -dehydrogenase: evidence for inverted substrate orientation (wrong-way binding) at the active site

Journal Article · Tue Jun 14 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00412a036· OSTI ID:6998231

Murdock, G L; Warren, J C; Sweet, F

Human placental estradiol 17..beta..-dehydrogenase was affinity labeled with 17lambda-estradiol 17-(bromo(2-/sup 14/C)acetate) (10 ..mu..M) or 17..beta..-estradiol 17-(bromo(2-/sup 14/C)acetate) (10 ..mu..M). The steroid bromoacetates competitively inhibit the enzyme (against 17..beta..-estradiol) with K/sub i/ values of 90 ..mu..M (17..cap alpha.. bromoacetate) and 134 ..mu..M(17..beta.. bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with t/sub 1/2/ = 110 min (17..cap alpha.. bromoacetate) and t/sub 1/2/ = 220 min (17..beta.. bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N/sup ..pi../-(carboxy(/sup 14/C)methyl histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N/sup ..pi../-(carboxy(/sup 14/C)methyl)histidine. Both the 17..cap alpha.. bromoacetate and also the 17..beta.. bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(..pi..CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involve in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17..cap alpha.. bromoacetate and 17..beta.. bromoacetate D-ring analogue of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone. Moreover, as each of the estradiol 17-(bromoacetates) undergoes the reversible binding step at the enzyme active site, its D-ring is in a reversed binding position relative to that of the natural substrate 17..beta..-estradiol as it undergoes catalytic hydrogen transfer at the same active site.

• Research Organization: Washington Univ. School of Medicine, St. Louis, MO (USA)
• OSTI ID: 6998231
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:12; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ANIMALS
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBOXYLIC ACID SALTS
ENZYMES
ESTRADIOL
ESTRANES
ESTROGENS
FEMALES
FETAL MEMBRANES
HEMIACETAL DEHYDROGENASES
HORMONES
HYDROXY COMPOUNDS
INACTIVATION
KINETICS
LABELLED COMPOUNDS
MAMMALS
MAN
MEMBRANES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PLACENTA
PRIMATES
REACTION KINETICS
STEROID HORMONES
STEROIDS
SUBSTRATES
VERTEBRATES
WOMEN