Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.     

人胎盘谷胱甘肽S-转移酶动力学及抑制剂的研究

研究了人胎盘型谷胱甘肽S-转移酶(GST-π)的动力学。底物GSH和1-氯-2,4-二硝基苯(CDNB)的km分别为0.109和0.870mmol/L。苯唑青霉素和先锋霉素Ⅰ能抑制GST—π,以先锋霉素较明显,属非竞争性抑制。溴磺酜对CDNB也是非竞争作用,但胆红素则对CDNB竞争而对GSH非竞争地抑制酶活力。S-正辛烷和S-正已烷谷胱甘肽与GSH竞争而与CDNB非竞争地抑制GST-π。已充分证明GST-π所催化的双底物反应属随机顺序机制。化学修饰实验发现:巯基、胍基、氨基、羧基和吲哚基可能参与酶活性中心的组成。     

Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione-Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 degrees C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 +/- 4.99 EU/mg proteins, 0.7447 +/- 0.0007 mM, and 11436 min(-1) for CDNB, and 88.00 +/- 2.30 EU/mg proteins, 0.3257 +/- 0.0012 mM, and 477 min(-1) for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].     

Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.     

Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene

Oxidative stress is known to induce cholestasis, but the underlying mechanisms are poorly understood. In this study we have characterized the short-term effects of tert-butyl hydroperoxide (t-BOOH)- and 1-chloro-2,4-dinitrobenzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2). The effects of t-BOOH and CDNB on bile formation, tissue GSH levels and subcellular Mrp2 localization were studied in perfused rat liver. Both, t-BOOH (0.5 mM) and CDNB (0.1 mM) induced within 60 min a decrease of hepatic GSH levels by more than 90% and an almost complete cessation of bile flow. As revealed by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its appearance inside the hepatocytes in putative intracellular vesicles. On the other hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV), another canalicular protein, and of zonula occludens associated polypeptide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieval pattern. Both, t-BOOH and CDNB induced a rapid net K+ efflux from the liver and a significant decrease of liver cell hydration. We conclude that severe glutathione depletion induces cholestasis by a retrieval of Mrp2, but not of DPPIV from the canalicular membrane. The underlying mechanism is unclear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect.     

Kinetic properties of the rat intestinal microsomal 1-naphthol:UDP-glucuronosyl transferase. Inhibition by UDP and UDP-N-acetylglucosamine

The kinetic properties of the rat intestinal microsomal 1-naphthol:UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic constants: Vmax = 102 +/- 6 nmol/min per mg microsomal protein, Km (UDPGlcUA) = 1.26 +/- 0.10 mM, Km (1-naphthol) = 96 +/- 10 microM and Ki (1-naphthol) = 25 +/- 7 microM. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso-Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP-N-acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl2 and Triton X-100.     

A Mu-class glutathione S-transferase from gills of the marine shrimp Litopenaeus vannamei: purification and characterization

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S-transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione-agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of approximately 25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 micromol/(min mg), and the K(m) values for CDNB and GSH are very similar (390 and 335 microM, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver-Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment.     

Dissection of the catalytic mechanism of isozyme 4-4 of glutathione S-transferase with alternative substrates

The kinetic and chemical mechanism of isozyme 4-4 of rat liver glutathione (GSH) S-transferase was investigated by using several alternative peptide substrates including N-acetyl-GSH, gamma-L-glutamyl-L-cysteine (gamma-GluCys), N4-(malonyl-D-cysteinyl)-L-2,4-diaminobutyrate (retro-GSH), and N4-(N-acetyl-D-cysteinyl)-L-2,4-diaminobutyrate (decarboxylated retro-GSH). The enzyme, which is normally stereospecific in the addition of GSH to the oxirane carbon of R absolute configuration in arene oxide substrates, loses its stereospecificity toward phenanthrene 9,10-oxide with the retro peptide analogues, giving a 2:1 mixture of the S,S and R,R stereoisomeric 9,10-dihydro-9-(S-peptidyl)-10-hydroxyphenanthrenes. The analogues with normal peptide bonds (N-acetyl-GSH and gamma-GluCys) show normal stereospecific addition. The kinetic mechanism of the enzyme was investigated by using the alternative substrate diagnostic with several 4-substituted 1-chloro-2-nitrobenzenes and GSH, N-acetyl-GSH, and gamma-GluCys. Varying the concentration of electrophile vs the identity of the GSH analogue and the concentration of GSH vs the identity of the electrophile gave two sets of intersecting reciprocal plots, a result consistent with a random sequential kinetic mechanism. The pH profiles of kc and kc/Ksm [saturating GSH, variable 1-chloro-2,4-dinitrobenzene (1)] exhibit a dependence on a deprotonation in the enzyme-GSH-1 and enzyme-GSH complexes with molecular pKa's of 6.1 and 6.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.     

Microtubule disassembly and morphologic alterations induced by 1-chloro-2,4-dinitrobenzene, a substrate for glutathione S-transferase

1-chloro-2,4-dinitrobenzene (CDNB), a potent substrate for glutathione S-transferase, is known to rapidly deplete cellular glutathione (GSH) via conjugate formation. Treatment of quiescent 3T3 cells with 5 uM CDNB results in disassembly of microtubules (MT) within 1 hr as revealed by indirect immunofluorescence microscopy. In addition, CDNB treatment also induces dramatic morphologic alterations similar to those mediated by colchicine. Furthermore, taxol prevents both MT disassembly and morphologic changes normally occurring in CDNB as well as colchicine-treated cells. The mechanism of CDNB-mediated MT disassembly in vivo and its possible relationship to cellular GSH metabolism are under current studies.     

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 degrees C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 mM) and bromosulfophthalein (0.1 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 mM) and oxidized glutathione (10 mM) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.     

The inhibition characteristics of human placental glutathione S-transferase-π by tricyclic antidepressants: amitriptyline and clomipramine

Tricyclic antidepressants (TCAs) are the non-selective amine re-uptake inhibitors, well absorbed from small intestine, cross the blood-brain barrier, distributed in the brain, and are bound to glutathione S-transferase-π (GST-π). TCAs can pass through placenta, accumulate in utero baby, and cause congenital malformations. Thus, the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of GST-π with amitriptyline and clomipramine was investigated. The K (m) values for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) were found to be 0.16 ± 0.04 and 3.60 ± 1.67 mM, respectively. The V (m) values were varying according to the fixed substrate; [CDNB] fixed, 53 ± 3 and [GSH] fixed 182 ± 63 U/mg protein. At variable [GSH] and variable [CDNB], the k (cat) values of 7.0 × 10(6) and 1.42 × 10(7) s(-1) and the k (cat)/K (m) values of 4.38 × 10(10) and 3.94 × 10(9 )M(-1 )s(-1) were obtained, respectively. At fixed [CDNB] and variable [GSH], amitriptyline (K (s) = 0.16 ± 0.03 mM; α = 2.08; and K (i) = 1.75 ± 0.37 mM) and clomipramine (K (s) = 0.24 ± 0.05 mM; α = 1.57; and K (i) = 3.90 ± 2.26 mM) showed linear mixed-type inhibition whereas when the varied substrate is CDNB, amitriptyline (K (i) = 4.90 ± 0.68 mM) and clomipramine (K (i) = 3.37 ± 0.39 mM) inhibition were noncompetitive. The inhibition of GST-π by TCAs means the destruction of its protective role against toxic electrophiles. The effect of antidepressants on fetus will be much severe, thus, the antidepressant therapy of pregnant women should be done with caution.     

Regulation of ketogenesis. Mitochondrial acetyl-CoA acetyltransferase from rat liver: initial-rate kinetics in the presence of the product CoASH reveal intermediary plateau regions

The analysis of the initial-rate kinetics of the liver mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) in the direction of acetoacetyl-CoA synthesis under product inhibition was performed. 1. Acetyl-CoA acetyltransferase shows a hyperbolic response of reaction velocity to changes in acetyl-CoA concentrations with an apparent Km of 0.237 +/- 0.001 mM. 2. CoASH is a (non-competitive) product inhibitor with a Kis of 22.6 microM and shifts the apparent Km for acetyl-CoA to the physiological concentration of this substrate in mitochondria (S0.5 = 1.12 mM in the presence of 121 microM CoASH). 3. CoASH causes a transformation of the Michaelis-Menten kinetics into initial-rate kinetics with four intermediary plateau regions. 4. The product analogue desulpho-CoA triggers a negative cooperativity as to the dependence of the reaction velocity on the acetyl-CoA concentration. These product effects drastically desensitize the acetyl-CoA acetyltransferase in its reaction velocity response to the acetyl-CoA concentrations and simultaneously extend the substrate dependence range. Thus a control of acetoacetyl-CoA synthesis by the substrate is established over the physiological acetyl-CoA concentration range. We suggest that this control mechanism is the key in establishing the rates of ketogenesis.     

Glutathione S-transferase from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit M(r) 22,000 having an optimum catalytic activity at pH 6.5-7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for K(m)(GST) and K(m)(CDNB) of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 degrees C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins.     

The kinetic mechanism of the hypothalamic progesterone 5 alpha-reductase

The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5 alpha-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent Km (progesterone) = 95.4 +/- 4.5 nM; apparent Kia(NADPH) = 9.9 +/- 0.7 microM). The plot of 1/v vs 1/progesterone intersected on the ordinate which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent Kis = 4.3 +/- 1.3 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 31.9 +/- 1.4 microM and apparent Kii = 145.4 +/- 15.5 microM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.     

Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 A resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat(CDNB) for the reaction is more than 3 times greater than kcat(FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km(FDNB) is about 20 times less than Km(CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.     

Distribution and properties of glutathione S-transferase from T. infestans

The glutathione transferase from T. infestans is able to render aqueous metabolites when incubated in vitro with malathion, parathion and fenitrothion. It is a soluble enzyme present in every developmental stage and widely distributed in all insect organs. The purification procedure applied, consisting of fractionation with ammonium sulfate and Bio-Gel P-60 chromatography, gives an unique molecular form catalytically active using methyl iodide as substrate in polyacrylamide gel electrophoresis (PAGE). One of the most active substrates is the 1-chloro-2,4-dinitrobenzene (CDNB), with an activity maximum at pH 7.5 and at 45 degrees C temperature. Its activation energy calculated from an Arrhenius plot is 14,846 cal mol-1. The enzyme susceptibility to inhibition by thiol reagents shows three degrees of responses; slight, moderate or high, depending on the compounds used. The kinetics of the enzyme catalysed reaction with the purified fraction is complex, and resembles that reported for glutathione S-transferase A from rat liver, showing a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of GSH. In general, the properties of this insect enzyme are similar to those enzymes isolated from vertebrate organisms.     

Glutathione S-transferases in human prostate

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).     

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.