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W2057029503 abstract "The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k cat/K m) with Δ5-androstene-3,17-dione was determined as 5 × 106m−1 s−1, which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 × 108 based on the ratio between k cat and the rate constant for the nonenzymatic isomerization of Δ5-androstene-3,17-dione. Besides being high in absolute numbers, the k cat/K m value of GST A3-3 exceeds by a factor of ∼230 that of 3β-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Δ5-Δ4 double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones. The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k cat/K m) with Δ5-androstene-3,17-dione was determined as 5 × 106m−1 s−1, which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 × 108 based on the ratio between k cat and the rate constant for the nonenzymatic isomerization of Δ5-androstene-3,17-dione. Besides being high in absolute numbers, the k cat/K m value of GST A3-3 exceeds by a factor of ∼230 that of 3β-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Δ5-Δ4 double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones. androstene-3,17-dione glutathione transferase glutathione electrophilic substrate-binding site polymerase chain reaction pregnene-3,20-dione 1-chloro-2,4-dinitrobenzene The metabolic pathways of steroid hormone biosynthesis leading to compounds such as testosterone and progesterone start with cholesterol and proceed in multiple steps involving oxidation and isomerization reactions (1Montgomery R. Conway T.W. Spector A.A. Chappell D. Biochemistry: A Case-oriented Approach. 6th Ed. Mosby-Year Book, Inc., St. Louis1996: 587-618Google Scholar). In one of the chemical transformations, Δ5-androstene-3,17-dione (Δ5-AD)1isomerizes to Δ4-AD, the immediate precursor of testosterone. A steroid isomerase that catalyzes this double-bond isomerization with high efficiency was discovered originally in bacteria (2Talalay P. Wang V.S. Biochim. Biophys. Acta. 1955; 18: 300-301Crossref PubMed Scopus (92) Google Scholar, 3Kuliopulus A. Mildvan A.S. Shortle D. Talalay P. Biochemistry. 1989; 28: 149-159Crossref PubMed Scopus (155) Google Scholar). A similar efficient enzyme is not present in mammalian tissues, but some glutathione transferases (GSTs) have also been known since the 1970s to exhibit steroid isomerase activity (4Benson A.M. Talalay P. Keen J.H. Jakoby W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 158-162Crossref PubMed Scopus (156) Google Scholar). However, GSTs differ from the bacterial isomerase in that they use the tripeptide glutathione (GSH) as a cofactor. The soluble GSTs occur as an enzyme superfamily of dimeric proteins playing a prominent role in the inactivation of numerous toxic electrophiles of both exogenous and endogenous origins. The majority of these detoxication reactions involve conjugation of GSH with the toxin via nucleophilic attack on the electrophilic center in the second substrate. The GSTs are grouped into different classes primarily based on primary structure (5Mannervik B. Ålin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1076) Google Scholar, 6Mannervik B. Awasthi Y.C. Board P.G. Hayes J.D. DiIlio C. Ketterer B. Listowsky I. Morgenstern R. Muramatsu M. Pearson W.R. Pickett C.B. Sato K. Widersten M. Wolf C.R. Biochem. J. 1992; 282: 305-306Crossref PubMed Scopus (630) Google Scholar). Variations of the amino acid residues that make up the electrophilic substrate-binding site (H-site) in different isoenzymes provide the GST family with the ability to catalyze reactions toward a large number of structurally diverse substrates. For example, in the Alpha class GST A4-4 exhibits high catalytic efficiency with biologically relevant alkenal substrates (7Hubatsch I. Ridderström M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (312) Google Scholar), whereas GST A1-1 and GST A2-2 efficiently catalyze the reduction of fatty acid and phospholipid hydroperoxides (8Zhao T.J. Singhal S.S. Piper J.T. Cheng J.Z. Pandya U. Clark-Wronski J. Awasthi S. Awasthi Y.C. Arch. Biochem. Biophys. 1999; 367: 216-224Crossref PubMed Scopus (108) Google Scholar). The isomerization of endogenous steroids differs from the ordinary type of GST-catalyzed reaction, because the GSH molecule serves as a base in the mechanism (9Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and is not consumed in the process. We have isolated a novel Alpha class glutathione transferase, GST A3-3, and discovered that it catalyzes essential Δ5-Δ4 isomerizations of intermediates in steroid hormone biosynthesis much more efficiently than any previously known GST or other human enzyme. The gene encoding GST A3-3 has been published (10Suzuki T. Johnston P.N. Board P.G. Genomics. 1993; 18: 680-686Crossref PubMed Scopus (40) Google Scholar), but isolation of the corresponding complete cDNA has not been reported previously, and evidence for the expression of the protein was lacking. Oligonucleotides that specifically would amplify the cDNA of GST A3-3 were designed based on the sequence of the GSTA3 gene (10Suzuki T. Johnston P.N. Board P.G. Genomics. 1993; 18: 680-686Crossref PubMed Scopus (40) Google Scholar). These primers, 5′GSTA3NCEcoRIForw and 3′GSTA3NCBamHIRev with the sequences 5′-ATATGAATTCAACCTCCAGAAGACTGTTACCATGGC-3′ and 5′-AATAATGGATCCTTCTTAGCCTCCATGGCTGCT-3′, respectively (restriction sites are underlined), were purchased from Interactiva (Ulm, Germany). The GSTA3 cDNA was amplified in a standard PCR using cloned Pfu DNA polymerase (Stratagene, La Jolla, CA) and Gene PoolTM cDNA derived from normal human placenta (Invitrogen, Groningen, The Netherlands) as template. The PCR conditions were: 94 °C for 5 min and 94 °C for 1 min, 54 °C for 2 min, and 72 °C for 2 min repeated 35 times followed by 72 °C for 7 min. The PCR product was purified from agarose gel using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) and cloned into the cloning vector pGEM®-3Z (Promega, Madison, WI) using the EcoRI and BamHI restriction sites. The cDNA was propagated in Escherichia coli XL1-Blue (Stratagene) and subjected to sequencing on both strands using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (USB Corporation, Cleveland, OH). Gene PoolTMcDNA derived from human testis, ovaries, and adrenal gland, purchased from Invitrogen, and cDNA libraries derived from human liver, thymus, skeletal muscle, and fetal brain provided generously by Dr. Peter Gustavsson (Department of Genetics and Pathology, Uppsala University) were used as templates in GSTA3-specific PCRs. Gene PoolTM cDNA from placenta was used as template in a positive control reaction, and PCRs using primers specific for glyceraldehyde-3-phosphate dehydrogenase were run in parallel as a quality control of the cDNA libraries. For each primer pair, a negative control reaction lacking cDNA was also performed. The isolated cDNAs from all GSTA3-expressing tissues were cloned and sequenced. An expression clone of GST A3-3 was constructed in several steps. The cDNA was PCR-amplified using cloned Pfu DNA polymerase and the primers GSTA33′NCBamHIRev (described above) and GSTA35′EcoRI (5′-AATAATGAATTCATGGCAGGGAAGCCAAGCTT-3′), which introduces an EcoRI restriction site (underlined) just before the start codon. The PCR product was digested with EcoRI andBamHI and cloned into pGEM®-3Z. Because of the presence of many internal restriction sites in the GSTA3 cDNA, we eliminated an internal SalI site by introducing a silent mutation to be able to subclone the cDNA into the expression vector pKK-D (11Björnestedt R. Widersten M. Board P.G. Mannervik B. Biochem. J. 1992; 282: 505-510Crossref PubMed Scopus (52) Google Scholar) using EcoRI and SalI. This was accomplished by using inverted PCR, cloned Pfu DNA polymerase, and the 5′-end phosphorylated primers GSTA3327Rev (5′-GCAGAAGAAGGATCATTTCATTCAAAT-3′) and GSTA3328Forw (5′-CCTTATGTCGTCCTGAGAAAAAGAT-3′) and pGEMGSTA3 as template. The following PCR program was used: 95 °C for 10 min followed by the cycle 95 °C for 1 min, 54 °C for 1 min, and 72 °C for 10 min repeated 35 times. Finally, the PCR product was extended at 72 °C for 30 min. The PCR product was subcloned into the expression vector pKK-D. The resulting expression clone named pKK-DGSTA3 was sequenced in its entirety to verify that no unwanted mutations had been introduced. E. coli XL-1 Blue carrying the expression clone pKK-DGSTA3 was grown in 2× TY (1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl) at 37 °C overnight. The culture was diluted 200 times in 2× TY and grown to an A 600 of 0.35, and the protein expression was induced by the addition of isopropyl-β-d-thiogalactoside to a final concentration of 0.2 mm. After 16 h of growth, the cells were harvested followed by lysing using ultrasonication. GST A3-3 was purified from the lysate by affinity chromatography (12Simons P.C. Vander Jagt D.L. Anal. Biochem. 1977; 82: 334-341Crossref PubMed Scopus (440) Google Scholar) using glutathione-Sepharose (Amersham Pharmacia Biotech). GST A3-3 was eluted with buffer containing 10 mm GSH. The purity was determined using SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining and amino acid analysis after hydrolysis. The extinction coefficient 23,900 ± 540 m−1cm−1 at 280 nm for the protein subunit was determined on the basis of the absorption spectrum and the results of the amino acid analysis. The specific activities of GST A3-3 for the isomerization reactions with Δ5-AD and Δ5-PD, purchased from Steraloids, Inc. (Newport, RI), were determined at 248 nm with 1.0 mm GSH, pH 8.0 (25 mm sodium phosphate buffer), at 30 °C. Extinction coefficients used were 16.3 mm−1cm−1 for Δ4-AD (4Benson A.M. Talalay P. Keen J.H. Jakoby W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 158-162Crossref PubMed Scopus (156) Google Scholar) and 17.0 mm−1 cm−1 for Δ4-PD (13Thomas J.L. Berko E.A. Faustino A. Myers R.P. Strickler R.C. J. Steroid Biochem. Mol. Biol. 1988; 31: 785-793Crossref Scopus (98) Google Scholar). The specific activity of GST A3-3 toward a panel of substrates comprised of 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, cumene hydroperoxide, and 2-cyano-1,3-dimethyl-1-nitrosoguanidine, commonly employed for characterizing GSTs, was determined by spectrophotometric assays at 30 °C under standard conditions (14Mannervik B. Widersten M. Pacifici G.M. Fracchia G.N. Advances in Drug Metabolism in Man. European Commission, Luxembourg1995: 407-459Google Scholar). In addition, the activity was measured with nonenal (7Hubatsch I. Ridderström M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (312) Google Scholar) and the isothiocyanates phenethylisothiocyanate and sulforaphane (15Meyer D.J. Crease D.J. Ketterer B. Biochem. J. 1995; 306: 565-569Crossref PubMed Scopus (97) Google Scholar,16Kolm R.H. Danielson U.H. Zhang Y. Talalay P. Mannervik B. Biochem. J. 1995; 311: 453-459Crossref PubMed Scopus (239) Google Scholar). The isomerization activity of GST A3-3 with Δ5-AD and Δ5-PD was monitored both at pH 7.4 in PBS (37 °C) and pH 8.0 in 25 mm sodium phosphate buffer (30 °C) at the saturating 1 mm concentration of GSH. The concentration of Δ5-AD was varied between 0.5 and 100 µm, and the concentration of Δ5-PD ranged between 2 and 20 µm. (It was not possible to use higher concentrations of Δ5-PD because of its limited solubility.) For comparison, the activity of GST A1-1 (17Stenberg G. Björnestedt R. Mannervik B. Protein Expression Purif. 1992; 3: 80-84Crossref PubMed Scopus (60) Google Scholar) with Δ5-AD was measured at pH 7.4 in PBS at 37 °C. The conjugation activity with CDNB was determined in 0.1 m sodium phosphate, pH 6.5, at 30 °C in the presence of 5 mm GSH and by varying the concentration of CDNB between 25 and 1.5 mm. Data points were also collected at a constant concentration of Δ5-AD (200 µm) or CDNB (1.5 mm) at concentrations of GSH between 0.020 and 1.5 mm at 30 °C, pH 8.0 and pH 6.5, respectively. The steady-state kinetic parameters were determined by fitting the Michaelis-Menten equation to the data points using Prism 2.0 (GraphPad, San Diego, CA). The inhibitory effect of the product Δ4-AD (50 µm) was measured at 1 mm GSH and by varying the concentration of Δ5-AD between 5 and 100 µm. TheK i value of Δ4-AD was determined by fitting the equation for competitive inhibition to the experimental data. cDNA libraries from various tissues were screened for GSTA3 expression using GSTA3-specific PCR. GSTA3 cDNA comprising 719 base pairs was amplified from the four tissues, placenta, ovary, adrenal gland, and testis. No expression of GST A3-3 could be detected in liver, thymus, skeletal muscle, or fetal brain (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase-specific PCR, run in parallel, amplified glyceraldehyde-3-phosphate dehydrogenase sequences from all cDNA libraries, showing that all the cDNA libraries used were of high quality (results not shown). Sequencing of both strands of the isolated GSTA3 cDNA from the four different tissues derived from four different individuals demonstrated that the nucleotide sequences were identical. However, the isolated GSTA3 cDNA differed from the previously published partial cDNA (18Board P.G. Biochem. J. 1998; 330: 827-831Crossref PubMed Scopus (56) Google Scholar) in one position. C instead of A at nucleotide position 208 in the partial GSTA3 cDNA translates into a Leu instead of an Ile at residue 70 in the protein. Comparison with the sequence of the exons in the GSTA3 gene (10Suzuki T. Johnston P.N. Board P.G. Genomics. 1993; 18: 680-686Crossref PubMed Scopus (40) Google Scholar) showed a difference at six nucleotide positions, of which four result in differences in the amino acid sequence. Because the GSTA3 cDNA sequences isolated in the present study differ from the previously published partial cDNA (18Board P.G. Biochem. J. 1998; 330: 827-831Crossref PubMed Scopus (56) Google Scholar) in only one position, it is likely that most of the nucleotide differences as compared with the sequence of the GSTA3 gene are caused by errors in the analysis of the GSTA3 gene and not to a genetic polymorphism. Whether the sequence variation in codon 70 is caused by a true polymorphism needs to be investigated further. In addition to the amplified full-length GSTA3 cDNA, a 52-base pair shorter PCR product was obtained in all GSTA3-positive tissues (Fig.1). Sequencing demonstrated that the shorter sequences were identical in all tissues and that it differed from the full-length cDNA only in length because of the absence of exon 3 in the shorter cDNA. The GSTA3 transcript lacking exon 3, if translated, would give rise to a truncated protein consisting of 32 amino acids, because joining the 3′ end of exon 2 with the 5′ end of exon 4 results in a +1 frameshift. A different truncated transcript of the GSTA3 gene was isolated previously (18Board P.G. Biochem. J. 1998; 330: 827-831Crossref PubMed Scopus (56) Google Scholar). In this, a stretch of 26 nucleotides was attached to the 5′ end of exon 3. The isolation of several distinct GSTA3 transcripts suggests that the pre-mRNA of GSTA3 is subject to alternative splicing. GST A3-3 was purified successfully from an E. coli expression culture. A single band on SDS-polyacrylamide gel electrophoresis and the results of a total amino acid analysis showed that the full-length protein was expressed. The finding that the enzyme could be stored on ice for several months without losing activity shows that the enzyme is stable. Polyclonal anti-GST A1-1 antibodies (19Hao X.-Y. Castro V.M. Bergh J. Sundström B. Mannervik B. Biochim. Biophys. Acta. 1994; 1225: 223-230Crossref PubMed Scopus (41) Google Scholar) cross-reacted with purified GST A3-3 in a Western blot and produced a single band, indicating that GST A3-3 possesses a structure with epitopes similar to those of GST A1-1. The isoelectric point of GST A3-3 was determined as 9.3, which is the highest value determined for any cloned human Alpha class GST (7Hubatsch I. Ridderström M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (312) Google Scholar,20Stockman P.K. McLellan L.I. Hayes J.D. Biochem. J. 1987; 244: 55-61Crossref PubMed Scopus (45) Google Scholar). GST A3-3 displays the highest isomerase activity with the steroid substrates Δ5-AD and Δ5-PD measured for any of the known Alpha class GSTs (TableI).Table ISpecific activities of recombinant human GST A3–3 with different electrophilic substrates compared with activities of other human class Alpha GSTsElectrophile (mm)GSHSpecific activityGST A3–3GST A1–1GST A2–2GST A4–4mmµmol mg−1 min−1Steroid substratesΔ5-Androstene-3,17-dione (0.1)1.0197 ± 15401-aRef. 9.0.21-aRef. 9.0.031-aRef. 9.Δ5-Pregnene-3,20-dione (0.01)1.037 ± 23.2 ± 0.1Other GST substratesCDNB (1.0)1.023 ± 2801-bRef. 14.801-bRef. 14.7.51-cRef. 7.Nonenal (0.1)0.52.2 ± 0.20.8 ± 0.12051-cRef. 7.Ethacrynic acid (0.2)0.250.17 ± 0.010.21-bRef. 14.0.11-bRef. 14.1.91-cRef. 7.Phenethylisothiocyanate (0.1)1.04.1 ± 0.71.71-dRef. 16.0.21-cRef. 7.Sulphoraphane (0.4)1.04.0 ± 0.31.91-dRef. 16.Cumene hydroperoxide (1.5)1.02.6 ± 0.2101-eRef. 21.101-eRef. 21.11-cRef. 7.Cyano-1,3-dimethyl-1-nitrosoguanidine (1.0)1.0ND1-fND, not detectable.0.11-cRef. 7.1-a Ref. 9Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar.1-b Ref. 14Mannervik B. Widersten M. Pacifici G.M. Fracchia G.N. Advances in Drug Metabolism in Man. European Commission, Luxembourg1995: 407-459Google Scholar.1-c Ref. 7Hubatsch I. Ridderström M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (312) Google Scholar.1-d Ref. 16Kolm R.H. Danielson U.H. Zhang Y. Talalay P. Mannervik B. Biochem. J. 1995; 311: 453-459Crossref PubMed Scopus (239) Google Scholar.1-e Ref. 21Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3254) Google Scholar.1-f ND, not detectable. Open table in a new tab The specific activity with Δ5-AD was higher than with Δ5-PD partly because of a lower concentration of the latter substrate (caused by lower solubility) in the assay system. With other established GST substrates, the activity of GST A3-3 did not differ notably from that of GST A1-1. It is noteworthy that the specific activities of GST A3-3 with the other substrates were generally lower than those obtained with the steroids.K mGSH was determined to 110 ± 22 µm using either Δ5-AD or CDNB as second substrate, which is close to the K mGSHvalue determined for GST A1-1 (22Stenberg G. Board P.G. Carlberg I. Mannervik B. Biochem. J. 1991; 274: 549-555Crossref PubMed Scopus (49) Google Scholar). In tissues, GST A3-3 is therefore saturated with GSH, because intracellular GSH concentrations are in the millimolar range. Steady-state kinetic parameters of GST A3-3 with the steroid substrates and CDNB at 30 °C are given in TableII. The rate constant (k non) for the nonenzymatic isomerization of Δ5-AD is 1.7 × 10−7 s−1(23Hawkinson D.C. Eames T.C.M. Pollack R.M. Biochemistry. 1991; 30: 10849-10858Crossref PubMed Scopus (103) Google Scholar), and the enzymatic rate acceleration (k cat/k non) is consequently ∼6 × 108. To obtain values for direct comparison of GST A3-3 with the 3β-hydroxysteroid dehydrogenase/isomerase, catalytic activities were also determined in PBS at 37 °C (Table II, Fig. 3). The K i value of Δ4-AD, tested as a product inhibitor, was determined as 25 µm, which is not significantly different from theK m value of Δ5-AD (24 ± 4 µm). This K m value is approximately half of the K m value of GST A1-1 for Δ5-AD (K m = 49 ± 4 µm), indicating a higher affinity of GST A3-3 for the same substrate.Table IISteady-state kinetic parameters of recombinant human GST A3–3Substrate (conditions)k cat2-aActivity with Δ5-androstene-3,17-dione was measured at 1.0 mmGSH and at concentrations of Δ5-androstene-3,17-dione varying between 0.5 and 100 µm. Parameters for Δ5-pregnene-3,20-dione were determined at 1.0 mmGSH, and the concentration of Δ5-pregnene-3,20-dione ranged between 2 and 20 µm. The reactions with Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione were started by the addition of enzyme. Parameters for CDNB were determined at pH 6.5 and 30 °C using 5 mm GSH and by varying the concentration of CDNB between 0.025 and 1.5 mm. The k cat values are calculated per enzyme subunit.K m values refer to the substrate of varied concentration.K m2-aActivity with Δ5-androstene-3,17-dione was measured at 1.0 mmGSH and at concentrations of Δ5-androstene-3,17-dione varying between 0.5 and 100 µm. Parameters for Δ5-pregnene-3,20-dione were determined at 1.0 mmGSH, and the concentration of Δ5-pregnene-3,20-dione ranged between 2 and 20 µm. The reactions with Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione were started by the addition of enzyme. Parameters for CDNB were determined at pH 6.5 and 30 °C using 5 mm GSH and by varying the concentration of CDNB between 0.025 and 1.5 mm. The k cat values are calculated per enzyme subunit.K m values refer to the substrate of varied concentration.k cat/K ms−1µmmM−1s−1Δ5-Androstene-3,17-dione (pH 8.0, 30 °C)102 ± 1124 ± 44300 ± 400Δ5-Androstene-3,17-dione2-bMeasurements were performed in PBS, pH 7.4, at 37 °C.(pH 7.4, 37 °C)120 ± 424 ± 25000 ± 400Δ5-Pregnene-3,20-dione (pH 8.0, 30 °C)27 ± 317 ± 31600 ± 110Δ5-Pregnene-3,20-dione2-bMeasurements were performed in PBS, pH 7.4, at 37 °C.(pH 7.4, 37 °C)29 ± 417 ± 41700 ± 200CDNB (pH 6.5, 30 °C)22 ± 1200 ± 20110 ± 102-a Activity with Δ5-androstene-3,17-dione was measured at 1.0 mmGSH and at concentrations of Δ5-androstene-3,17-dione varying between 0.5 and 100 µm. Parameters for Δ5-pregnene-3,20-dione were determined at 1.0 mmGSH, and the concentration of Δ5-pregnene-3,20-dione ranged between 2 and 20 µm. The reactions with Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione were started by the addition of enzyme. Parameters for CDNB were determined at pH 6.5 and 30 °C using 5 mm GSH and by varying the concentration of CDNB between 0.025 and 1.5 mm. The k cat values are calculated per enzyme subunit.K m values refer to the substrate of varied concentration.2-b Measurements were performed in PBS, pH 7.4, at 37 °C. Open table in a new tab The metabolic pathways that lead from cholesterol to steroid hormones such as testosterone and progesterone proceed in multiple steps via the intermediate 3β-hydroxy-5-pregnene-20-one (1Montgomery R. Conway T.W. Spector A.A. Chappell D. Biochemistry: A Case-oriented Approach. 6th Ed. Mosby-Year Book, Inc., St. Louis1996: 587-618Google Scholar). This intermediate is transformed by oxidation of the 3β-hydroxy group followed by an obligatory isomerization of the resulting 3-keto-Δ5product into the 3-keto-Δ4 isomer (Fig.2). Oxidation and isomerization are common features of three alternative pathways distinguished by the processing of different portions of the pregnenone molecule (1Montgomery R. Conway T.W. Spector A.A. Chappell D. Biochemistry: A Case-oriented Approach. 6th Ed. Mosby-Year Book, Inc., St. Louis1996: 587-618Google Scholar). For example, dehydroepiandrosterone through oxidation forms Δ5-AD, which isomerizes to Δ4-AD, the immediate precursor of testosterone. Similarly, the isomerization of Δ5-PD to Δ4-PD is on the pathway to progesterone. The isomerization of 3-keto-Δ5 steroids to the Δ4 isomers is thermodynamically favored, but the nonenzymatic reaction is negligible under physiological conditions. The 3-keto compound is formed in a pyridine nucleotide-dependent 3β-hydroxysteroid dehydrogenase reaction (24Samuels L.T. Helmreich M.L. Lasater M.B. Reich H. Science. 1951; 113: 490-491Crossref PubMed Scopus (105) Google Scholar). The dehydrogenase has an associated steroid isomerase activity and is generally assumed to effect the isomerization of the primary product (1Montgomery R. Conway T.W. Spector A.A. Chappell D. Biochemistry: A Case-oriented Approach. 6th Ed. Mosby-Year Book, Inc., St. Louis1996: 587-618Google Scholar, 25Thomas J.L. Myers R.P. Strickler R.C. J. Steroid Biochem. Mol. Biol. 1989; 33: 209-217Crossref Scopus (133) Google Scholar). However, the catalytic efficiency (k cat/K m) of GST A3-3 with Δ5-AD is 230 times higher than the isomerase activity of the dehydrogenase (Fig. 3). At identical protein concentrations the isomerase activity of the “bifunctional” dehydrogenase is therefore negligible in comparison with the activity of GST A3-3. It should be noted that GSTs from other classes have significantly lower specific activities, and the high activities are characteristic of Alpha class isoenzymes (14Mannervik B. Widersten M. Pacifici G.M. Fracchia G.N. Advances in Drug Metabolism in Man. European Commission, Luxembourg1995: 407-459Google Scholar). GST A1-1 was previously the enzyme with the highest steroid isomerase activity known in human tissues (9Pettersson P.L. Mannervik B. J. Biol. Chem. 2001; 276: 11698-11704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), but we find that the catalytic efficiency of GST A3-3 is ∼20 times higher with both Δ5-AD and Δ5-PD (Fig. 3). The k cat/K m values of GST A3-3 for the steroid substrates (5.0 × 106 and 1.7 × 106m−1s−1) are in the range of 106–108m−1 s−1, characterizing highly efficient enzymes acting on their natural substrates (26Fersht A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W.H. Freeman & Co., New York1999Google Scholar). The catalytic efficiency with Δ5-AD displayed by GST A3-3 is one of the highest reported for a GST with any substrate, suggesting that it is of physiological importance. To play a role in steroid hormone biosynthesis, GST A3-3 has to be expressed in steroidogenic tissues. We have demonstrated by GST A3-specific PCR that testis, ovary, adrenal gland, and placenta contain GST A3-3 mRNA. All these tissues are characterized by the active production of steroid hormones. In contrast, thymus, liver, skeletal muscle, and fetal brain did not express any detectable GST A3-3 message (Fig. 1). GST A1-1 is a major hepatic enzyme (2–3% of the total cytosolic protein) and is also found in significant amounts in kidney and testis (27Rowe J.D. Nieves E. Listowsky I. Biochem. J. 1997; 325: 481-486Crossref PubMed Scopus (200) Google Scholar, 28Sherratt P.J. Pulford D.J. Harrison D.J. Green T. Hayes J.D. Biochem. J. 1997; 326: 837-846Crossref PubMed Scopus (141) Google Scholar, 29van Ommen B. Bogaards J.J.P. Peters W.H.M. Blaauboer B. van Bladeren P.J. Biochem. J. 1990; 269: 609-613Crossref PubMed Scopus (114) Google Scholar). However, GST A1-1 does not show the selective expression in steroidogenic tissues noted for GST A3-3. In human gonads, Alpha class GSTs have been immunohistochemically identified in cells relevant to hormone production, i.e.testicular interstitial Leydig cells and ovarian cells of the Graffian follicle and corpus luteum as well as cells in the reticular layer of the adrenal cortex (30Campbell J.A.H. Bass N.M. Kirsch R.E. Cancer. 1980; 45: 503-510Crossref PubMed Scopus (36) Google Scholar). A similar Alpha class-specific distribution was found in rat gonads (31Fukai F. Ohtaki H. Ueda T. Katayama T. J. Clin. Biochem. Nutr. 1992; 12: 93-107Crossref Scopus (10) Google Scholar). Although the polyclonal antibodies used do not distinguish GST A3-3 from other Alpha class enzymes, this selective tissue expression provides support for a role of GSTs in steroid hormone biosynthesis. What is the structural basis behind the marked differences between the Alpha class GSTs in isomerization activity with the steroid substrates? A protein sequence alignment of the human Alpha class GSTs is shown in Fig. 4. The three-dimensional structure of human GST A1-1 has been determined, and the residues that form the GSH-binding site (G-site, highlighted in blue) and the H-site (highlighted in red) have been identified (32Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (415) Google Scholar). A comparison of GST A3-3, GST A2-2, and GST A1-1 shows that the residues that make up the GSH-binding site are strictly conserved. 20 amino acids of 222 (including the initiator methionine) differ between GST A1-1 and GST A3-3 (91% identity); three are positioned in the H-site (Fig. 4). Five of the 26 residues that differ between GST A2-2 and GST A3-3 (88% identity) are situated in the H-site. The specific activity of GST A3-3 with Δ5-AD is 5- and 1000-fold higher than those of GST A1-1 and GST A2-2, respectively, despite the high sequence identity between these enzymes (Table I). This is a new example of how relatively small structural differences involving a few amino acid residues in Alpha class GSTs can exert a major effect on substrate specificity and catalytic efficiency. The introduction of a limited number of GST A4-4 residues into GST A1-1 has been shown previously to be enough for installing the high activity with alkenals that is characteristic of GST A4-4 (33Nilsson L.O. Gustafsson A. Mannervik B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9408-9412Crossref PubMed Scopus (68) Google Scholar). However, the sequence identity between GST A3-3 and GST A4-4 is low (54%), and the specific activity of GST A3-3 with Δ5-AD is 6500 times higher than that of GSTA4-4. The high activity with well established metabolic precursors and the selective expression of GST A3-3 in steroidogenic tissues strongly suggest that GST A3-3 has evolved specifically to participate in the biosynthesis of steroid hormones. Previously, the soluble GSTs have been regarded mainly as detoxication enzymes. Membrane-associated GSTs and related proteins have been linked to the metabolism of eicosanoids such as leukotrienes and prostaglandins (34Jakobsson P.J. Thorén S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (895) Google Scholar). However, several of the soluble enzymes such as GST A2-2, GST M2-2, and GST M3-3 also display activity of physiological relevance with prostaglandins (35Burgess J.R. Chow N.-W.I. Reddy C.C. Tu C.-P.D. Biochem. Biophys. Res. Commun. 1989; 158: 497-502Crossref PubMed Scopus (22) Google Scholar, 36Beuckmann C.T. Fujimori K. Urade Y. Hayaishi O. Neurochem. Res. 2000; 25: 733-738Crossref PubMed Scopus (80) Google Scholar). It should be noted also that 4-hydroxynonenal, which is a characteristic substrate for GST A4-4 (7Hubatsch I. Ridderström M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (312) Google Scholar), has chemotactic properties (37Müller K. Hardwick S.J. Marchant C.E. Law N.S. Waeg G. Esterbauer H. Carpenter K.L.H. Mitchinson M.J. FEBS Lett. 1996; 388: 165-168Crossref PubMed Scopus (58) Google Scholar) and activates the epithelial growth factor receptor (38Suc I. Meilhac O. Lajoie-Mazenc I. Vandaele J. Jürgens G. Salvayre R. Negre-Salvayre A. FASEB J. 1998; 12: 665-671Crossref PubMed Scopus (132) Google Scholar). Thus, the steroid isomerase activity of GST A3-3 adds to the emerging paradigm of GSTs being linked to various facets of biological signaling in addition to their role in cellular defense against electrophiles. We thank Dr. Kristina Lagerstedt at the Department of Genetics and Pathology (Uppsala University) for useful help and advice and Dr. Peter Gustavsson of the same department for generously providing cDNA libraries." @default.
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