Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.

The kinetic mechanism catalysed by homogeneous rat liver 3 alpha-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs.

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3α-HSD catalyzes the inactivation of androgens in the prostate by converting 5α-dihydrotestosterone to 3α-androstanediol, where excess 5α-dihydrotestosterone is implicated in prostate disease. By contrast, 20α-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20α-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3α-HSDs and 20α-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3α-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3α-HSD was converted to 20α-HSD by replacing these loops with those found in 20α-HSD. However, when pocket residues in 3α-HSD were mutated to those found in 20α-HSD altered specificity was not achieved. Replacement of loop A created a 17β-HSD activity that was absent in either 3α- or 20α-HSD. Once loops A and C were replaced, the chimera had both 3α- and 20α-HSD activity. When loops A, B, and C were substituted, 3α-HSD was converted to a stereospecific 20α-HSD with a resultant shift in kcat/Km for the desired reaction of 2 × 1011. This study represents an example where sex hormone specificity can be changed at the enzyme level.

https://www.pnas.org/content/pnas/96/20/11161.full.pdf

Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins.

The enzyme, rat ovarian 20α-hydroxysteroid dehydrogenase (20αHSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20α-hydroxypregn-4-ene-3-one (20α-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20αHSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20αHSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20αHSD expressed as glutathione-S-transferase-20αHSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatogr...

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Expression, Purification and Characterization of the Rat Luteal 20α-Hydroxysteroid Dehydrogenase1

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity The final enzyme preparation was purified 252-fold, with a recovery of 14% Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000 The isoelectric point was approximately pH 61 The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 63 The apparent Km for NADH was 81 microM with cortisone (50 microM) as the cosubstrate Substrate inhibition was observed with concentrations of NADH greater than 01 mM The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 79 The apparent Km for NAD+ was 526 microM The initial reaction velocities with NADPH were less than 50% of those with NADH The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase

Piamsook Pongsawasdi

Papers

Kinetic studies of rat ovarian 20α-hydroxysteroid dehydrogenase

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides☆

Studies of the coenzyme binding site of rat ovarian 20α-hydroxysteroid dehydrogenase☆