Abstract
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.
Highlights
The human type 1 and type 2 isoforms of 3-hydroxysteroid dehydrogenase/isomerase (3HSD1 and 3-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern
Our primary focus has been on determining the structural basis for the 14 –16-fold higher affinity of purified human 3-HSD1 for substrate and inhibitor steroids compared with human 3HSD2
There is a strict tissue-specific distribution of the two human 3-HSD isoforms. 3-HSD1 is expressed in mammary gland, breast tumors, prostate, prostate tumors, and placenta, and 3-HSD2 is expressed in the adrenals, testes, and ovaries
Summary
Materials—Dehydroepiandrosterone and pyridine nucleotides were purchased from Sigma; 5-androstene-3,17-dione was from Steraloids Inc. (Newport, RI); reagent grade salts, chemicals, and analytical grade solvents were from Fisher. Kinetic constants for the isomerase substrate were determined at 27 °C in incubations of 5-androstene-3,17dione (20 –100 M), with or without NADH (0.05 mM) and purified enzyme (0.01– 0.04 mg) in 0.02 M potassium phosphate buffer, pH 7.4. Kinetic constants for the 3-HSD cofactor were determined for the purified mutant and wild-type enzymes in incubations containing NADϩ (10 –100 M), dehydroepiandrosterone (100 M), and purified enzyme (0.04 mg) in 0.02 M potassium phosphate, pH 7.4, at 27 °C using the spectrophotometric assay at 340 nm. For Q240R1 and 3-HSD1, the incubations at 27 °C contained subsaturating concentrations of dehydroepiandrosterone (4.0 and 8.0 M, relative to substrate Km values of 3.7– 6.1 M), epostane or trilostane (0 –1.0 M), NADϩ (0.1 mM), and purified human type 1 enzyme (0.03– 0.04 mg) in 0.02 M potassium phosphate buffer, pH 7.4. The modeled images were produced using the Ribbons 2.0 program [18]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have