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Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2.

Publication ,  Journal Article
Thomas, JL; Boswell, EL; Scaccia, LA; Pletnev, V; Umland, TC
Published in: J Biol Chem
June 3, 2005

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

June 3, 2005

Volume

280

Issue

22

Start / End Page

21321 / 21328

Location

United States

Related Subject Headings

  • Tissue Distribution
  • Substrate Specificity
  • Structure-Activity Relationship
  • Sequence Homology, Amino Acid
  • Protein Conformation
  • Protein Binding
  • NAD
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
 

Citation

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Thomas, J. L., Boswell, E. L., Scaccia, L. A., Pletnev, V., & Umland, T. C. (2005). Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2. J Biol Chem, 280(22), 21321–21328. https://doi.org/10.1074/jbc.M501269200
Thomas, James L., Elizabeth L. Boswell, Launa A. Scaccia, Vladimir Pletnev, and Timothy C. Umland. “Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2.J Biol Chem 280, no. 22 (June 3, 2005): 21321–28. https://doi.org/10.1074/jbc.M501269200.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

June 3, 2005

Volume

280

Issue

22

Start / End Page

21321 / 21328

Location

United States

Related Subject Headings

  • Tissue Distribution
  • Substrate Specificity
  • Structure-Activity Relationship
  • Sequence Homology, Amino Acid
  • Protein Conformation
  • Protein Binding
  • NAD
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data