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

Abstract

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3␤-hydroxysteroid dehydrogenase/isomerase (3␤-HSD1 and 3␤-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His 156 contributes to the 14-fold higher affinity that 3␤-HSD1 exhibits for substrate and inhibitor steroids compared with human 3␤-HSD2 containing Tyr 156 in the otherwise identical catalytic domain. Our structural model of human 3␤-HSD localizes His 156 or Tyr 156 in the subunit interface of the enzyme homodimer. The model predicts that Gln 105 on one enzyme subunit has a higher probability of interacting with His 156 on the other subunit in 3␤-HSD1 than with Tyr 156 in 3␤-HSD2. The Q105M mutant of 3␤-HSD1 (Q105M1) shifts the Michaelis-Menten constant (K m ) for 3␤-HSD substrate and inhibition constants (K i ) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3␤-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3␤-HSD2. Our model also predicts that Gln 240 in 3␤-HSD1 and Arg 240 in 3␤-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 K m by 2.2-fold to a value similar to that of 3␤-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 3␤-HSD1. Thus, key structural reasons for the substantially higher affinities of 3␤-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 3␤-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.The human type 1 (placenta, mammary gland, and prostate) and type 2 (adrenals, ovary, and testis) isoforms of 3␤-hydroxysteroid dehydrogenase (EC 1.1.1.145)/steroid ⌬ 5 -⌬ 4 -isomerase (EC 5.3.3.1) (3␤-HSD1 1 and 3␤-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern (1). As shown in Fig. 1, human 3␤-HSD1 catalyzes the conversion of 3␤-hydroxy-5-ene-steroids (dehydroepiandrosterone (DHEA) and pregnenolone) to 3-oxo-4-ene-steroids (androstenedione and progesterone), and human 3␤-HSD2 converts 17␣-hydroxypregnenolone and pregnenolone to ultimately produce cortisol and aldosterone in the human adrenal, respectively (2). 17␣-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 17␤-hydroxysteroid dehydrogenase (17␤-HSD) to estradiol, which participates in the cascade of events that initiates labor in humans (2, 3). Placental 3␤-HSD1 also converts pregnenolone to...

A critical role for the histidine residues in the catalytic function of acyl‐CoA:cholesterol acyltransferase catalysis: Evidence for catalytic difference between ACAT1 and ACAT2

“….SF and FYxDWWN motifs [10] are identified by green boxes and blue boxes, respectively. that His156 of 3bHSD1 interacts with the 3b-hydroxyl group of the steroid substrate with 14-fold higher affinity compared with 3bHSD2 containing Tyr156 [35]. Substrate binding is not impaired substantially in all histidine mutants and, on the basis of this feature, it is likely that critical histidine residues of ACAT isoenzymes are not involved in the stabilization of the transition-state intermediate.…”

Rational proteomics V: Structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3β-hydroxysteroid dehydrogenase/isomerase type 1

“…6) due to Gln105Met and His156Tyr mutations [8,35] was demonstrated in a dramatic increase in the substrate K m and inhibitor K i values of 3β-HSD_1 to equal those measured for 3β-HSD_2. In the dimer model, the side chain of Gln105 forms an intersubunit H-bond with the side chain of Arg93.…”

“…In an alternate conformation it has the potential to form an inter-monomer H-bond with the side chain of His156. This H-bond is disrupted in 3β-HSD_1 by the replacement of Gln105 with Met and His156 with Tyr, which is the residue present in 3β-HSD_2 [35].…”

Structure/function of the inhibition of human 3β-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

“…Our structural model also predicts that the Thr187 of 3β-HSD2 is positioned to interact with the carbonyl group of the nicotinamide moiety of enzyme-bound NAD + , while Met187 of 3β-HSD1 is unlikely to interact with this functional group on cofactor. Met187/Thr187 is the only different amino acid in 3β-HSD1 and 3β-HSD2 in the region of the enzyme active-site that approximates the nicotinamide group of NAD + and the A-ring of bound steroid [5,6], suggesting that this may be a key structural basis for the high affinity of 3β-HSD1 for the inhibitors and substrates relative to 3β-HSD2. To test this prediction, the M187T mutation of 3β-HSD1 has been created, expressed and purified for kinetic analyses of enzyme inhibition by trilostane and 4α,5α-epoxy-testosterone as well as for analyses of substrate and cofactor kinetics.…”

“…However, His156/Tyr156 was shown to reside in the α-helical subunit interface of the dimeric enzyme. This residue does not interact directly with bound substrate or inhibitor steroids but apparently influences subunit interactions that modify the topography of the active site [6]. Identifying fingerprint residues that interact with key groups on inhibitors, substrates and cofactors may determine the structural basis of the higher affinity of 3β-HSD1 for ligands relative to 3β-HSD2.…”