Residue 234 in glutathione transferase T1-1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates (original) (raw)
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Archives of Biochemistry and Biophysics, 1997
forms toward anti-CDE was investigated by molecular modeling of the two proteins with GSH conjuga-The kinetics of the conjugation of glutathione tion products in their active sites. These studies re-(GSH) with anti-1,2-dihydroxy-3,4-oxy-1,2,3,4-tetravealed that the enantioselectivity of hGSTP1-1 for hydrochrysene (anti-CDE), the activated form of the (/)-anti-CDE and the differential catalytic efficiencwidespread environmental pollutant chrysene, cataies of the V104 and I104 forms of hGSTP1-1 in the lyzed by two naturally occurring polymorphic forms GSH conjugation of (/)-anti-CDE were due to the difof the pi class human GSH S-transferase (hGSTP1ferences in the active-site architecture of the two 1), has been investigated. The polymorphic forms of proteins. The results of the present study, for the hGSTP1-1, which differ in their primary structure by first time, provide evidence for the toxicological relea single amino acid in position 104, exhibited prefervance of GSTP1-1 polymorphism in humans and sugence for the GSH conjugation of (/)-anti-CDE, which gest that the population polymorphism of hGSTP1-1 is a far more potent carcinogen than (0)-anti-CDE. variants with disparate enzyme activities may, at When concentration of anti-CDE was varied (5-200 least in part, account for the differential susceptibil-mM) and the GSH concentration was kept constant ity of individuals to environmental carcinogens such at 2 mM, both hGSTP1-1(I104) and hGSTP1-1(V104) as anti-CDE and possibly other similar carcinogens. obeyed Michaelis-Menten kinetics. However, the ᭧ 1997 Academic Press V max of GSH conjugation of anti-CDE was approxi-Key Words: Chrysene; carcinogenesis; glutathione Smately 5.3-fold higher for the V104 variant than for transferase P1-1; polymorphism; detoxification. the I104 form. Calculation of catalytic efficiency (k cat / K m) thus resulted in a value for hGSTP1-1(V104), 28 mM 01 s 01 , that was 7.0-fold higher than that for hGSTP1-1(I104), 4 mM 01 s 01. The mechanism of the GSTs 3 belong to a superfamily of multifunctional isodifferences in the kinetic properties of hGSTP1-1 isoenzymes which can detoxify a wide variety of electrophilic xenobiotics primarily by catalyzing their conju-1 This investigation was supported, in part, by United States Public Health Service Grants CA 55589 (S.V.S.), CA 63660 (S.A.), ES 07804 (P.Z.), and GM 32304 (Y.C.A.) and by the National Cancer 3 Abbreviations used: (/)-anti-CDE, chrysene-1R,2S-diol 3S,4R-Institute, Department of Health and Human Services, under contract with ABL (X.J.). The contents of this publication do not necessarily oxide; (0)-anti-CDE, chrysene-1S,2R-diol 3R,4S-oxide; (/)-anti-BPDE, benzo(a)pyrene-7R,8S-diol 9S,10R-oxide; CDNB, 1-chloro-reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or 2,4-dinitrobenzene; GSH, glutathione; GST, glutathione S-transferase; H-site, electrophilic xenobiotic binding site; PAHs, polycyclic organizations imply endorsement by the United States Government. 2 To whom correspondence may be addressed. aromatic hydrocarbons.
Biochemistry, 2004
The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the R class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.
Archives of Biochemistry and Biophysics, 1997
whose pK a value thus is lowered by 3 pH units by the enzyme. Three differences in the cDNA as compared Recombinant human theta class glutathione transto the sequence previously published were found. One ferase T1-1 has been heterologously expressed in Eschof these differences causes a change in the deduced erichia coli and a simple purification method involvamino acid sequence and involves the nucleotide triping immobilized ferric ion affinity chromatography let encoding amino acid 126, which was determined as and Orange A dye chromatography is described. The GAG (Glu), instead of the published GGG (Gly). ᭧ 1997 catalytic properties of the enzyme differ significantly Academic Press from those of other glutathione transferases, also Key Words: 1,2-epoxy-3-(4-nitrophenoxy)propane; within the theta class, with respect to both substrate EPNP; glutathione transferase; theta class glutathione selectivity and kinetic parameters. In addition to 1,2transferase; pH dependence; steady state kinetics; subepoxy-3-(4-nitrophenoxy)propane, the substrate used strate selectivity. previously to monitor the enzyme, human glutathione transferase T1-1 has activity with the naturally occurring phenethylisothiocyanate and also displays glutathione peroxidase activity with cumene hydro-Glutathione transferases (GSTs) 2 are detoxication peroxide. Further, the enzyme is active with 4-nitroenzymes capable of catalyzing the conjugation of glutabenzyl chloride and 4-nitrophenethyl bromide, but thione (GSH) with a wide variety of electrophilic comshows no detectable activity with the more chemically pounds [for recent reviews see (1, 2)]. Mammalian solureactive 1-chloro-2,4-dinitrobenzene. The Michaelis ble GSTs have been divided into six classes, alpha, constant for glutathione, K GSH m , with 1,2-epoxy-3-(4-nitrophenoxy)propane as second substrate, is high at kappa, mu, pi, sigma, and theta, based on sequence low pH values but decreases at higher pH values. This similarities (3-7). Theta class GSTs were identified by is mirrored in k cat /K GSH m which increases with an appar-Meyer et al. (4) and Hiratsuka et al. (8), although GST ent pK a value of 9.0, reflecting the ionization of the activity with menaphthyl sulfate, later shown to derive thiol group of glutathione in solution. The same results from rat glutathione transferase T2-2 (rGST T2-2) 3 was are obtained with 4-nitrophenethyl bromide as elecdetected 20 years earlier (10). Further, Fjellstedt et trophilic substrate, although the K GSH m value (0.72 mM al. (11) described the isolation of an enzyme that was at pH 7.5), as well as the pK a (8.1) derived from the pH probably rGST T1-1, since it displayed high activity dependence of k cat /K GSH m , are lower with this substrate. with 1,2-epoxy-3-(4-nitrophenoxy)propane and other In contrast, k cat and k cat /K electrophile m display either a maxepoxides. imum or a plateau at pH 7.0-7.5, and an apparent pK a Human theta class glutathione transferase T1-1, value of 5.7 was determined for the pH dependence of k cat with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates.
GLUTATHIONE S-TRANSFERASES: A BRIEF ON CLASSIFICATION AND GSTM1-T1 ACTIVITY
The glutathione S-transferase (GST) isoenzyme superfamilies detoxify a wide-range of toxic chemicals and environmental substances are extensively expressed in mammalian tissues. Liver and pancreas are the sites where cytosolic Phase I and phase II biotransformation GSTs enzymes have characteristic expression. GSTs play a key role in the deactivation of reactive oxygen species (ROS) and the metabolism of lipids, chemotherapeutic agents. GSTs are mainly involved in conjugation of reduced glutathione (GSH) with diverse substrates specificity and it is possible that genetic variations in these enzymes will influence cellular response to the environmental agents. GSTs are overexpressed in response to a chemical or oxidative stress as an adaptive physiology and upregulated in cancerous state of organ or tissue. GSTs are essentially involved in susceptibility to various forms of cancer as they are vital in detoxification mechanism to metabolize the environmental carcinogens. GSTM1 encodes for a class mu GST isoenzyme involved in polycyclic aromatic hydrocarbons (PAHs) detoxification. The substrates of GSTM1 include benzo(a)pyrene, benzo(c)phenanthrene, benzo(g)chrysene and other carcinogens. They can catalyze in-vitro GSH conjugation with several potent carcinogenic epoxides including aflatoxin B1(AFB1)8,9-epoxide and electrophilic metabolites of PAHs present in tobacco smoke. Ethylene dibromide, p-nitrobenzyl chloride, p-nitrophenetyl bromide, methyl chloride, and methyl iodide, are known substrates for GSTT1 or GST Theta (θ). GST Theta is most primitive among other known GSTs and widely expressed in nature.
1999
The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared. The theta class glutathione S-transferase (rGSTT1-1) from rat liver had an anity for dihalomethanes lower by three orders of magnitude (K app > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase. This provided new evidence that formaldehyde was not the main toxicant associated with GSTmediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed eects. The marked dierences in substrate anity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic eects associated with glutathionemediated catalysis of DCM conversion.
Biochemical and Biophysical Research Communications, 1997
kinetic properties and enantioselectivity of hGSTP1-1 In this study, we demonstrate that the active site variants toward anti-BPDE was investigated by modarchitecture of the human glutathione (GSH) S-transeling of the two proteins with conjugation product ferase Pi (GSTP1-1) accounts for its enantioselectivity molecules in their active sites. Molecular modeling in the GSH conjugation of 7b,8a-dihydroxy-9a,10astudies revealed that the differences in catalytic propoxy-7,8,9,10-tetrahydrobenzo(a) pyrene (anti-BPDE), erties of hGSTP1-1 variants as well as the enantioselecthe ultimate carcinogen of benzo(a)pyrene. Furthertivity of hGSTP1-1 in the GSH conjugation of antimore, we report that the two polymorphic forms of BPDE can be rationalized in terms of the architecture human GSTP1-1, differing in their primary structure of their active sites. Our results suggest that the popuby a single amino acid in position 104, have disparate lation polymorphism of hGSTP1-1 variants with dispaactivity toward (/)-anti-BPDE, which can also be rarate enzyme activities may, at least in part, account tionalized in terms of their active site structures. for the differential susceptibility of individuals to car-When concentration of (/)-anti-BPDE, which among cinogens such as anti-BPDE and possibly other similar four BPDE isomers is the most potent carcinogen, was carcinogens. ᭧ 1997 Academic Press varied and GSH concentration was kept constant at 2 mM (saturating concentration), both forms of hGSTP1-1 [hGSTP1-1(V104) and hGSTP1-1(I104)] obeyed Michaelis-Menten kinetics. The V max of GSH conjugation
Chemical Research in Toxicology, 2002
In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (-)-anti-and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (k cat /K m ) of 464 mM -1 s -1 with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (-)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high k cat /K m with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM -1 s -1 for (-)-anti-DBPDE]. Lowest k cat /K m value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM -1 s -1 was estimated for (+)-syn-DBPDE and 3.4 mM -1 s -1 with (-)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (-)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212fTyr212 and Ala216fVal216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to (-)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates. † Part of this study was presented at the 18th International
Carcinogenesis, 1998
Previous studies have identified allelic variants of the human glutathione transferase (GST) Pi gene and showed that the two different encoded proteins with isoleucine (GSTP1-1/I-105) or valine (GSTP1-1/V-105) at position 105, respectively, differ significantly in their catalytic activities with model substrates. Moreover, recent epidemiological studies have demonstrated that individuals differing in the expression of these allelic variants also differ in susceptibility to tumour formation in certain organs, including such in which polycyclic aromatic hydrocarbons (PAH) may be etiological factors. In the present study the catalytic efficiencies (k cat /K m ) of these GSTP1-1 variants were determined with a number of stereoisomeric bay-region diol epoxides, known as the ultimate mutagenic and carcinogenic metabolites of PAH, including those from chrysene, benzo[a]pyrene and dibenz[a,h]anthracene. In addition, GSTP1-1 mutants in which amino residue 105 is alanine (GSTP1-1/A-105) or tryptophan (GSTP1-1/W-105) have been constructed and characterized. GSTP1-1/V-105 was found to be more active than GSTP1-1/I-105 in conjugation reactions with the bulky diol epoxides of PAH, being up to 3-fold as active towards the anti-and syn-diol epoxide enantiomers with R-absolute configuration at the benzylic oxiranyl carbon. Comparing the four enzyme variants, GSTP1-1/A-105 generally demonstrated the highest k cat /K m value and GSTP1-1/W-105 the lowest with the anti-diol epoxides. A close correlation was observed between the volume occupied by the amino acid residue at position 105 and the value of k cat /K m . With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased k cat /K m values. The mutational replacement of isoleucine with alanine or tryptophan at position 105 did not alter the enantio selectivity of the GSTP1-1 variants compared with the naturally occurring allelic variants GSTP1-1/I-105 and GSTP1-1/V-105. Since the amino acid at position 105 forms part of the substrate binding site (H-site) the effect of increasing bulkiness is expected to cause restricted access of the diol epoxide and proper alignment of the two reactants for efficient glutathionyl-*Abbreviations: PAH, polycyclic aromatic hydrocarbons; CDNB, 1-chloro 2,4-dinitrobenzene; syn-and anti- CDE,4epoxide;h]anthracene-1,2-diol 3,4-epoxide.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2010
Background: The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site. Methods: Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants. Results: The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other. Conclusions: H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1. General significance: The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.