Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, two distinct isoenzymes of 11beta-hydroxysteroid-dehydrogenase (11beta-HSD) have been cloned and characterized in several species: The isoenzyme 11beta-HSD-I is widely distributed, bidirectional, prefers NADP(H) and has a low substrate affinity. The isoenzyme 11beta-HSD-II seems to exclusively oxidize physiological glucocorticoids, uses NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroids fluorinated in position 9, however, are rapidly reduced by human kidney cortex slices. We attempted to find out which isoenzyme is responsible for this unexpected reductase activity. We studied the 11beta-HSD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidney cortex. For the reaction E to F (not for DH-D to D!), glucose-6-phosphate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/NADPH-regenerating system. Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael's constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmax(oxidation)/Vmax(reduction) (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmax(oxidation)/Vmax(reduction) was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner. In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11beta-HSD-II. The NADH-dependent 11beta-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11beta-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11beta-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.
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PMID:Metabolism of dexamethasone in the human kidney: nicotinamide adenine dinucleotide-dependent 11beta-reduction. 914 56

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11beta-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11beta-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11beta-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11beta-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11beta-HSD and MR messenger RNAs (mRNAs), and for type I 11beta-HSD protein. Leydig cell 11beta-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11beta-HSD is present in rat Leydig cells. However, unlike the type I 11beta-HSD in liver parenchymal cells, the Leydig cell 11beta-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 +/- 9.3 nM and maximum velocity (Vmax) of 7.1 +/- 1.2 pmol x min x 10(6) cells. The second component consisted of high Km activities that were consistent with type I:NADP-dependent oxidative activity with Km of 5.87 +/- 0.46 microM and Vmax of 419 +/- 17 pmol x min x 10(6) cells, and NADPH-dependent reductive activity with Km of 0.892 +/- 0.051 microM and Vmax of 117 +/- 6 pmol x min x 10(6) cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11beta-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11beta-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.
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PMID:Identification of a kinetically distinct activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells. 916 33

In the human and in rodents like the rat and mouse, the liver enzyme 11 beta-hydroxysteroid dehydrogenase type I (11 beta-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11 beta-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular, mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11 beta-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10(-6) mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10(-3) mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with K(m) values of 0.64 mumol/l and 9.8 mumol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate K(m) was 7.2 mumol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar K(m) and Vmax values for NAD+ to NADP+ as cosubstrates (K(m) of 4.3 mumol/l and 5.0 mumol/l respectively). The NAD+ preference for the oxidation of 10(-6) mol/l cortisol described above may be due to a second, NAD(+)-preferring 11 beta-HSD with a K(m) of 1.4 mumol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (K(m): 1.2 mumol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11 beta-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD(+)-preferring. This kidney enzyme (analogous to 11 beta-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.
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PMID:Evidence for isoforms of 11 beta-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig. 916 19

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.213, AKR1C9) is a member of the aldo-keto reductase (AKR) superfamily which inactivates circulating steroid hormones. We have proposed a catalytic mechanism in which Tyr 55 acts as a general acid with its pK value being lowered by a hydrogen bond with Lys 84 which is salt-linked to Asp 50. To test this mechanism, residues at the active site were mutated and the mutant enzymes (Y55F, Y55S, K84M, K84R, D50N, D50E, and H117A) were purified to homogeneity from an Escherichia coli expression system. Spectrophotometric assays showed that mutations of Tyr 55 and Lys 84 gave enzymes that were apparently inactive for steroid oxidation and reduction. All mutants appeared inactive for steroid reduction. The catalytic efficiencies for steroid oxidation were reduced 4-10-fold for the Asp 50 mutants and 300-fold for the H117A mutant. Fluorescence titration with NADPH demonstrated that each mutant bound cofactor unimpeded. Equilibrium dialysis indicated that the competitive inhibitor testosterone formed E.NADH.testosterone complexes only with the Y55F, Y55S, and D50N mutants with Kd values 10-fold greater than those for wild-type. Therefore the loss of steroid oxidoreductase activity observed for the Tyr 55 mutants cannot be attributed simply to an inability to bind steroid. Using a highly sensitive radiometric assay in which the conversion of [14C]-5 alpha-dihydrotestosterone (DHT) to [14C]-3 alpha-androstanediol (3 alpha-Diol) was measured, the rate enhancement (kcat/knoncat) for the reaction was estimated to be 2.6 x 10(9). Using this assay, all mutants formed steroid product with decreases in an overall rate enhancement of 10(1)-10(4). It was found that Tyr 55 made the single largest contribution to rate enhancement. This is the first instance where point mutations in the conserved catalytic tetrad of an AKR yielded enzymes which were still catalytically active. This enabled the construction of pH vs kcat profiles for the reduction of [14C]-5 alpha-DHT catalyzed by the tetrad mutants. These profiles revealed that the titratable group assigned to the general acid (pK = 6.50 +/- 0.42) was eliminated in the Y55F and H117A mutants. The pH-independent value of kcat was decreased in the H117A and Y55F mutants, by 2 and 4 log units, respectively. pH vs kcat(app) profiles for the oxidation of [3H]-3 alpha-Diol showed that the same titratable group (pK = 7.50 +/- 0.30) was eliminated in both the Y55F and K84M mutants but was retained in the H117A mutant. Since only the Y55F mutant eliminated the titratable group in both the reduction and oxidation directions it is assigned as the catalytic general acid/base. The differential effects of His 117 and Lys 84 on the ionization of Tyr 55 are explained by a "push-pull" mechanism in which His 117 facilitates proton donation and Lys 84 facilitates proton removal by Tyr 55.
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PMID:Mutagenesis of 3 alpha-hydroxysteroid dehydrogenase reveals a "push-pull" mechanism for proton transfer in aldo-keto reductases. 952 75

In previous studies in human subjects metyrapone has been found to exert significant extra-adrenal effects, consistent with an effect on the 11-reductase activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). In the present study the effects of metyrapone on cortisone metabolism by rat liver microsomes were investigated. Aliquots of microsomal preparations were incubated with NADPH cofactor and different concentrations of cortisone for a range of time intervals up to 30 min. The products of the reaction were extracted with ethyl acetate and separated using thin-layer chromatograph. Cortisol was estimated by radioimmunoassay. There was a linear increase in cortisol formation over the first 150 sec of the reaction. Over this time period metyrapone had no effect on the rate of the reaction. When the reaction was allowed to proceed for 30 min, however, metyrapone caused a 50% decrease in the amount of cortisol formed. These data suggest that metyrapone may alter cortisone-cortisol conversion by directly interacting with 11beta-HSD but in this system metyrapone does not appear to have the characteristics of a conventional enzyme inhibitor.
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PMID:Effects of metyrapone on hepatic cortisone-cortisol conversion in the rat. 988 46

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.
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PMID:The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase. 1038 26

The serum concentration of active glucocorticosteroids depends not only on adrenal synthesis but also on enzymatic activation of 11-dehydro-glucocorticoids in the liver by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). In order to define the respective involvement of other regulative enzymes in the metabolism of 11-dehydro-glucocorticoids in the liver, the objective of this study was to evaluate the kinetic behavior of NADPH:delta 4-3-ketosteroid-5alpha-reductase (5alpha-reductase, EC 1.3.99.5). The interrelations to liver 11beta-HSD1 will be discussed. The kinetic properties of 5alpha-reductase of the rabbit liver were measured by a radioenzymatic assay and characterized with respect to protein-, substrate-, cosubstrate-, and pH-dependence. Michaelis-Menten enzyme kinetic parameters (Km and Vmax) were obtained for the formation of 5alpha-reduced 11-dehydrocorticosterone and corticosterone metabolites. We found that both 11-dehydrocorticosterone (Km 4.2 x 10(-6) mol/l, Vmax 2,600 pmol x min(-1) x mg(-1)) and corticosterone (Km 0.5 x 10(-6) mol/l, Vmax 38 pmol x min(-1) x mg(-1)) exhibit a high affinity to 5alpha-reductase. With respect to cosubstrate-, pH-dependence and finasteride inhibition, it is likely that 11-dehydrocorticosterone metabolism is primarily controlled by isoenzyme 5alpha-reductase type 1. This study shows that the deactivation of GCS especially of 11-dehydro-glucocorticoids via 5alpha-reductase is an important metabolic pathway in the liver. The metabolic activation of GCS by 11beta-HSD could possibly lead to an excess of GCS in the hepatocytes. Due to 5alpha-reductase activity this excess can be limited - on the level of CORT as well as of 11-DHC.
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PMID:Kinetic studies on rabbit liver glucocorticoid 5alpha-reductase. 1072 9

We studied 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1 and the effects of different steroids on them. Cortisol was oxidized in the presence of NAD as well as NADP, reflecting the presence of two different 11beta-HSD forms. Enzyme kinetics for cortisol 11beta-oxidation were: Vmax = 5.9 pmol/(min x mg), Km = 0.2 microM with NAD, and Vmax = 4.5 pmol/(min x mg), Km = 1.0 microM with NADP. Interestingly, no reverse reaction was observed when using cortisone and NADPH as substrate and cosubstrate, respectively. Exposure of cells to a variety of steroids had different effects on cortisol 11beta-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3-60 min of exposure) decreased the NAD-dependent 11beta-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11beta-oxidation of cortisol with NADP increased by dexamethasone treatment of the cells, after a lagtime of about 2 h, and this effect was still evident after 32 h. The increase of 11beta-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC50:125 nM). The antiglucocorticoid RU486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 microM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11beta-oxidation, but had no effect on the NAD-dependent 11beta-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11beta-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK1 cells, two forms of 11beta-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11beta-HSD 2, the NADP-dependent 11beta-HSD possibly resembles an as yet unknown third isoform.
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PMID:Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform? 1078 27

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in T-47D cells, while 11beta-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11beta-HSD catalytic activity was elevated 11-fold, while estrone (E(1)), estradiol (E(2)) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11beta-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11beta-HSD2 gene expression, increasing the steady-state levels of 11beta-HSD2 mRNA. Taken together, these results demonstrate that 11beta-HSD2 is the 11beta-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.
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PMID:Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells. 1082 13

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) regulate androgen and estrogen concentrations in mammals. By 1995, four distinct enzymes with 17beta-HSD activity had been identified--17beta-HSD-types 1 and 3, which, in vivo, are NADPH-dependent reductases; and 17beta-HSD-types 2 and 4, which, in vivo, are NAD(+)-dependent oxidases. Since then, six additional enzymes with 17beta-HSD activity have been isolated from mammals. With the exception of 17beta-HSD-type 5, which belongs to the aldoketo-reductase (AKR) family, these 17beta-HSDs belong to the short chain dehydrogenase/reductase (SDR) family. Several 17beta-HSDs appear to be examples of convergent evolution. That is, 17beta-HSD activity arose several times from different ancestors. Some 17beta-HSDs share a common ancestor with retinoid oxido-reductases and have retinol dehydrogenase activity. 17beta-HSD-types 2, 6 and 9 appear to have diverged from ancestral retinoid dehydrogenases early in the evolution of deuterostomes during the Cambrian, about 540 million years ago. This coincided with the origin of nuclear receptors for androgens and estrogens suggesting that expression of 17beta-HSDs had an important role in the early evolution of the physiological response to androgens and estrogens.
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PMID:Evolution of 17beta-hydroxysteroid dehydrogenases and their role in androgen, estrogen and retinoid action. 1116 32


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