Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple and sensitive method was developed for the quantification of serum total 3alpha-hydroxy bile acids. 0.1 ml of serum was mixed with tris(hydroxymethyl) aminomethane hydrochloric acid buffer and heated at 67 degrees C for 30 min. To the solution were added 3alpha-hydroxysteroid : oxidoreductase (EC 1.1.1.50; 3alpha-HSD), NAD, diaphorase (EC 1.6.4.3) and resazurin. The mixture was incubated at 20 degrees C for 1 h. The resultant fluorescence of resorfin was measured at 580 nm with the excitation at 560 nm. The blank value was obtained after the same treatment of another 0.1 ml of the same serum without 3alpha-HSD. A linear relationship was obtained between the amount of bile acids and the fluorescence intensities in the range of 1 to 150 mumol/1. The recovery of bile acids added to the serum was 81.4 +/- 2.5 (S.D.)% for cholate, chenodeoxycholate and deoxycholate. The bile acid content in the serum was 48.8 mumol/1 with a standard deviation of +/- 0.42 and a coefficient of variation of +/- 0.87% in 10 replicate determinations. The mean bile acid content of normal fasting male sera was 8.0 mumol/1 (3.6-12.6 mumol/1, n = 12) and of female sera 6.8 mumol/1 (3.2-12.7 mumol/1, n = 13).
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PMID:A simple and sensitive assay of total serum bile acids. 94 25

Aldo-keto reductases (AKR) are monomeric oxidoreductases that retain a conserved catalytic tetrad (Tyr, Lys, Asp, and His) at their active sites in which the Tyr acts as a general acid-base catalyst. In rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9), a well-characterized AKR, the catalytic tyrosine is Tyr 55. This enzyme displays a high catalytic efficiency for a common AKR substrate 9,10-phenanthrenequinone (9,10-PQ). Surprisingly, Y55F and Y55S mutants of 3alpha-HSD reduced 9,10-PQ with high kcat values. This is the first report whereby the invariant catalytic tyrosine of an AKR has been mutated with retention of kcat values similar to wild-type enzyme. The Y55F and Y55S mutants displayed narrow substrate specificity and reduced select aromatic quinones and alpha-dicarbonyls. kcat versus pH profiles for steroid oxidoreduction catalyzed by wild-type 3alpha-HSD exhibited a single ionizable group with a pK= 7.0-7.5, which has been assigned to Tyr 55. This group was not evident in the kcat versus pH profiles for 9, 10-PQ reduction catalyzed by either wild-type or the Tyr 55 mutant enzymes, indicating that the protonation state of Tyr 55 is unimportant for 9,10-PQ turnover. Instead, wild-type and the active-site mutants Y55F, Y55S, H117A, D50N, K84R, and K84M showed the presence of a new titratable group with a pKb = 8.3-9.9. Thus, the group being titrated is not part of the tetrad. All the mutants decreased kcat/Km considerably more than they decreased kcat. Thus, the K84R mutant demonstrated a 30-fold decrease in the pH-independent value of kcat but 2200-fold decrease in the pH-independent value of kcat/Km. This suggests that all the tetrad residues influence quinone binding and that Lys 84 plays a dominant role in maintaining proper substrate orientation. Using wild-type enzyme, the energy of activation (Ea) for 9,10-PQ reduction was approximately 11 kcal/mol less than steroid oxidoreduction. The Ea for 9,10-PQ reduction was unchanged in the Tyr 55 mutants, suggesting that the reaction proceeds through the same low-energy barrier in the wild-type enzyme and these mutants. The retention of quinone reductase activity in this AKR in the absence of Tyr 55 with kcat versus pH rate profiles and activation energies identical to wild-type enzyme suggests that quinone reduction occurs via a mechanism that differs from 3-ketosteroid reduction. In this mechanism, the electron donor (NADPH) and acceptor (o-quinone) are bound in close proximity, which permits hydride transfer without formal protonation of the acceptor carbonyl by Tyr 55. This represents a rare example where one enzyme can catalyze the same chemical reaction (carbonyl reduction) by either acid catalysis or by a propinquity effect and where these two mechanisms can be discriminated by site-directed mutagenesis.
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PMID:Retention of NADPH-linked quinone reductase activity in an aldo-keto reductase following mutation of the catalytic tyrosine. 969 94

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3alpha-HSDs, Type 5 17beta-HSD, ovarian 20alpha-HSDs as well as the steroid 5beta-reductases. Selective inhibitors of 3alpha-HSD isoforms could control occupancy of the androgen and GABA(A) receptors, while broader based AKR inhibitors targeting 3alpha-HSD, 20alpha-HSD and prostaglandin F2alpha synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3alpha-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP-), and the ternary complex (E.NADP+.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad (Tyr55, Lys84, His117 and Asp50) participates in a 'proton-relay' in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the pKb and pKa, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5beta-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.
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PMID:Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase. 1041 95

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.
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PMID:Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase. 1530 46