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Exploring the Functional Plasticity of Human Glutathione Transferases: Allelic Variants, Novel Isoenzyme and Enzyme Redesign
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic centra. Members of this family have also been implicated in different facets of biological signaling.

The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme stability and activity with various substrates was investigated. A valine instead of an isoleucine in position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105 gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105 increase the thermal stability of the enzyme.

GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well.

The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, reactions taking place in the biosynthesis of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine 9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond isomerase activity. A leucine in position 111 appears to have a major role in productive binding of the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity.

GST A2-2 is a poor catalyst of the steroid double-bond isomerization of Δ5-androstene-3,17-dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a highly efficient double-bond isomerase by mutating five active-site residues to the corresponding residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a rational approach to redesign of these enzymes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , p. 56
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 695
Keywords [en]
Biochemistry
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-1858ISBN: 91-554-5270-1 (print)OAI: oai:DiVA.org:uu-1858DiVA, id: diva2:161428
Public defence
2002-04-19, B42, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-03-27 Created: 2002-03-27Bibliographically approved
List of papers
1. Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105
Open this publication in new window or tab >>Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105
1998 (English)In: Journal of Molecular Cell Biology, ISSN 1674-2788, E-ISSN 1759-4685, Vol. 278, no 3, p. 687-698Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89714 (URN)10.1006/jmbi.1998.1708 (DOI)9600848 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
2. Differences in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons
Open this publication in new window or tab >>Differences in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons
Show others...
1998 (English)In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 19, no 3, p. 433-436Article in journal (Refereed) Published
Abstract [en]

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 (kcat/Km) 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 kcat/Km 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 kcat/Km. With the syn-diol epoxides, such a correlation was observed with alanine, valine and isoleucine, whereas tryptophan was associated with increased kcat/Km 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 glutathionylation. In conclusion, the present study indicates that individuals who are homozygous for the allele GSTP1* B (coding for GSTP1-1/V-105) display a higher susceptibility to malignancy because of other factors than a decreased catalytic efficiency of GSTP1-1/V-105 in the detoxication of carcinogenic diol epoxides of benzo[a]pyrene or structurally related PAH.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89715 (URN)9525277 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
3. The human glutathione transferase P1-1 specific inhibitor TER 117 designed for overcoming cytostatic-drug resistance is also a strong inhibitor of glyoxalase I
Open this publication in new window or tab >>The human glutathione transferase P1-1 specific inhibitor TER 117 designed for overcoming cytostatic-drug resistance is also a strong inhibitor of glyoxalase I
2000 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 57, no 3, p. 619-624Article in journal (Refereed) Published
Abstract [en]

gamma-L-Glutamyl-S-(benzyl)-L-cysteinyl-R-(-)-phenylglycine (TER 117) has previously been developed for selective inhibition of human glutathione S-transferase P1-1 (GST P1-1) based on the postulated contribution of this isoenzyme to the development of drug resistance in cancer cells. In the present investigation, the inhibitory effect of TER 117 on the human glyoxalase system was studied. Although designed as an inhibitor specific for GST P1-1, TER 117 also competitively inhibits glyoxalase I (K(I) = 0.56 microM). In contrast, no inhibition of glyoxalase II was detected. Reduced glyoxalase activity is expected to raise intracellular levels of toxic 2-oxoaldehydes otherwise eliminated by glyoxalase I. The resulting toxicity would accompany the potentiation of cytostatic drugs, caused by inhibition of the detoxication effected by GST P1-1. TER 117 was designed for efficient inhibition of the most abundant form GST P1-1/Ile105. Therefore, the inhibitory effect of TER 117 on a second allelic variant GST P1-1/Val105 was also studied. TER 117 was shown to competitively inhibit both GST P1-1 variants. The apparent K(I) values at glutathione concentrations relevant to the intracellular milieu were in the micromolar range for both enzyme forms. Extrapolation to free enzyme produced K(I) values of approximately 0.1 microM for both isoenzymes, reflecting the high affinity of GST P1-1 for the inhibitor. Thus, the allelic variation in position 105 of GST P1-1 does not affect the inhibitory potency of TER 117. The inhibitory effects of TER 117 on GST P1-1 and glyoxalase I activities may act in synergy in the cell and improve the effectiveness of chemotherapy.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89716 (URN)10692504 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
4. Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones
Open this publication in new window or tab >>Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones
2001 In: Journal of Biological Chemistry, Vol. 276, no 35, p. 33061-33065Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89717 (URN)
Available from: 2002-03-27 Created: 2002-03-27Bibliographically approved
5. Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3
Open this publication in new window or tab >>Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 19, p. 16648-16654Article in journal (Refereed) Published
Abstract [en]

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89718 (URN)10.1074/jbc.M201062200 (DOI)11872752 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved
6. Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase
Open this publication in new window or tab >>Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, p. 30019-30022Article in journal (Refereed) Published
Abstract [en]

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89719 (URN)10.1074/jbc.M204485200 (DOI)12023294 (PubMedID)
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2017-12-14Bibliographically approved

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