Identification of Key Amino Acids Responsible for the Substantially Higher Affinities of Human Type 1 3β-Hydroxysteroid Dehydrogenase/Isomerase (3β-HSD1) for Substrates, Coenzymes, and Inhibitors Relative to Human 3β-HSD2
Link: jbc.org/content/280/22/21321.full#F7
LOOK CLOSELY AT NADH. NADH is an enzyme broken down from Vitamin B3. You can buy NADH at any nutrional store. It’s a bit expensive…However, this may increase 3-hsd activity…
Abstract
The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD1 and 3β-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 3β-HSD1 exhibits for substrate and inhibitor steroids compared with human 3β-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3β-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 3β-HSD1 than with Tyr156 in 3β-HSD2. The Q105M mutant of 3β-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3β-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3β-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3β-HSD2. Our model also predicts that Gln240 in 3β-HSD1 and Arg240 in 3β-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 3β-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 3β-HSD1. Thus, key structural reasons for the substantially higher affinities of 3β-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 3β-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.
Previous SectionNext SectionThe human type 1 (placenta, mammary gland, and prostate) and type 2 (adrenals, ovary, and testis) isoforms of 3β-hydroxysteroid dehydrogenase (EC 1.1.1.145)/steroid Δ5-Δ4-isomerase (EC 5.3.3.1) (3β-HSD11 and 3β-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern (1). As shown in Fig. 1, human 3β-HSD1 catalyzes the conversion of 3β-hydroxy-5-ene-steroids (dehydroepiandrosterone (DHEA) and pregnenolone) to 3-oxo-4-ene-steroids (androstenedione and progesterone), and human 3β-HSD2 converts 17α-hydroxypregnenolone and pregnenolone to ultimately produce cortisol and aldosterone in the human adrenal, respectively (2). 17α-Hydroxylase/17,20 lyase (CYP17) in the human adrenal gland converts pregnenolone to DHEA, which is the major circulating steroid in humans as DHEA sulfate (2). In placenta, androstenedione is converted by aromatase and 17β-hydroxysteroid dehydrogenase (17β-HSD) to estradiol, which participates in the cascade of events that initiates labor in humans (2, 3). Placental 3β-HSD1 also converts pregnenolone to progesterone to help maintain the uterus in a quiescent state throughout human pregnancy (3). In addition to placenta and other human peripheral tissues, the 3β-HSD1 is selectively expressed in breast tumors (4) and prostate tumors (5, 6), where it catalyzes the first step in the conversion of circulating DHEA to estradiol or testosterone to promote tumor growth. Determination of the structure/function relationships of human 3β-HSD1 and 3β-HSD2 may lead to the development of highly specific inhibitors of 3β-HSD1 that can help control the timing of labor and slow the growth of hormone-sensitive tumors without inhibiting 3β-HSD2, so that steroidogenesis in the adrenal gland to produce cortisol and aldosterone is not compromised.
The two-step reaction of 3β-HSD/isomerase using DHEA as substrate is shown in Fig. 2. This reaction scheme shows the reduction of NAD+ to NADH by the rate-limiting 3β-HSD activity and the requirement of this NADH for the activation of isomerase on the same enzyme protein. Because the isomerase reaction is irreversible, the 3β-HSD/isomerase cannot convert androstenedione to DHEA (7, 8). According to our stopped-flow fluorescence spectroscopy study, NADH induces a time-dependent conformational change in the enzyme structure as the isomerase activity reaches a maximum over 1 min after the addition of the coenzyme (9). The intermediate steroid, 5-androstene-3,17-dione, remains bound during the reaction sequence (7, 9). Our homology model (10) of human 3β-HSD structure (Fig. 3) predicts that a difference in the amino acid sequence at position 240 (Gln in 3β-HSD1 and Arg in 3β-HSD2) may be responsible for the 3-fold higher affinities of 3β-HSD1 for isomerase substrate, NAD+, and NADH compared with 3β-HSD2. The current study tests this prediction using site-directed mutagenesis.
