RESUMO
Human GSTP1-1 is one of the most important proteins, which overexpresses in a large number of human tumours and is involved in the development of resistance to several anticancer drugs. So, it has become an important target in cancer treatment. In this study, 12 benzothiazole derivatives were synthesized and screened for their in vitro inhibitory activity for hGSTP1-1. Among these compounds, two of them (compounds #2 and #5) have been found to be the leads when compared with the reference drug etoposide. In order to analyse the structure-activity relationships (SARs) and to investigate the binding side interactions of the observed lead compounds, a HipHop pharmacophore model was generated and the molecular docking studies were performed by using CDocker method. In conclusion, it is observed that the lead compounds #2 and #5 possessed inhibitory activity on the hGSTP1-1 by binding to the H-site as a substrate in which the para position of the phenyl ring of the benzamide moiety on the benzothiazole ring is important. Substitution at this position with a hydrophobic group that reduces the electron density at the phenyl ring is required for the interaction with the H side active residue Tyr108.
Assuntos
Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , Benzotiazóis/síntese química , Inibidores Enzimáticos/síntese química , Humanos , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-AtividadeRESUMO
The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.
Assuntos
Ácidos Carboxílicos/química , Ácido Glutâmico/química , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Glutationa/química , Glutationa/metabolismo , Sítios de Ligação/genética , Catálise , Dinitroclorobenzeno/química , Dinitroclorobenzeno/metabolismo , Glutationa/análogos & derivados , Glutationa Transferase/genética , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica/genética , Especificidade por Substrato/genética , Tirosina/química , Tirosina/genéticaRESUMO
Glutathione transferase A4-4 (GST A4-4) is involved in the detoxication of lipid peroxidation products such as alkenals. The human enzyme has been heterologously expressed in Escherichia coli, but for more extensive characterization of the enzyme the expression level had to be elevated. A clone providing up to 8-fold higher yields was created, by screening an expression library with random silent mutations in the 5' region of the cDNA encoding GST A4-4.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/biossíntese , Códon , DNA Complementar/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Glutationa Transferase/genética , Humanos , Mutagênese , Proteínas Recombinantes/biossínteseRESUMO
Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase. We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.
Assuntos
Antimaláricos/metabolismo , Artemisininas , Glutationa Transferase/metabolismo , Sesquiterpenos/metabolismo , Animais , Derivados de Benzeno/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa S-Transferase pi , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Rim/enzimologia , Fígado/enzimologia , Masculino , NADP/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMO
Glutathione transferases comprise a large family of cellular detoxification enzymes that function by catalyzing the conjugation of glutathione (GSH) to electron-deficient centers on carcinogens and other toxins. NMR methods have been used to characterize the structure and dynamics of a human class pi enzyme, GST P1-1, in solution. Resonance assignments have been obtained for the unliganded enzyme and the GSH and S-hexylglutathione (GS-hexyl) complexes. Differences in chemical shifts between the GSH and GS-hexyl complexes suggest more extensive structural differences between these two enzyme-ligand complexes than detected by previous crystallographic methods. The NMR studies reported here clearly show that an alpha-helix (alpha2) within the GSH binding site exists in multiple conformations at physiological temperatures in the absence of ligand. A single conformation of alpha2 is induced by the presence of either GSH or GS-hexyl or a reduction in temperature to below 290 K. The large enthalpy of the transition ( approximately 150 kJ/mol) suggests a considerable structural rearrangement of the protein. The Gibbs free energy for the transition to the unfolded form is on the order of -4 to -6 kJ/mol at physiological temperatures (37 degrees C). This order-to-disorder transition contributes substantially to the overall thermodynamics of ligand binding and should be considered in the design of selective inhibitors of class pi glutathione transferases.
Assuntos
Glutationa Transferase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Glutationa Transferase/química , Humanos , Ligantes , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.
Assuntos
Glutationa Transferase/metabolismo , Sequência de Bases , Catálise , Primers do DNA , Glutationa Transferase/genética , Humanos , Isoenzimas , Cinética , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
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 Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-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 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-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 approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) 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.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Esteroides/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli , Biblioteca Gênica , Glutationa Transferase/genética , Hormônios/biossíntese , Humanos , Isoenzimas/genética , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esteroide Isomerases/metabolismo , Especificidade por SubstratoRESUMO
An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.
Assuntos
Sequência Conservada , Glutationa Transferase/metabolismo , Motivos de Aminoácidos , Ativação Enzimática , Estabilidade Enzimática , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de ProteínaRESUMO
Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pK(a) of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pK(a) 7.1. In both cases the pK(a) was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pK(a) values > or = 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Sequência de Bases , Domínio Catalítico/genética , Primers do DNA/genética , Glutationa Transferase/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Tirosina/química , Água/químicaRESUMO
Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.
Assuntos
Androstenodiona/metabolismo , Glutationa Transferase/metabolismo , Glutationa/fisiologia , Androstenodiona/química , Androstenodiona/fisiologia , Ligação Competitiva , Catálise , Glutationa Transferase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Isoenzimas , Isomerismo , CinéticaRESUMO
The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.
Assuntos
Lactoilglutationa Liase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Sítios de Ligação , Primers do DNA , Cinética , Lactoilglutationa Liase/química , Lactoilglutationa Liase/genética , Modelos Moleculares , Mutagênese , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
There are multiple glutathione transferase genes, the proteins for which have different substrate specificities. The various genes are differentially expressed such that species and organs and tissues differ qualitatively and quantitatively for cytosolic and membrane-bound forms. This unit provides protocols for analysis of transferase activity in a continuous spectrophotometric assay and an assay with dichloromethane as the substrate.
Assuntos
Glutationa Transferase/metabolismo , Humanos , Especificidade por SubstratoRESUMO
Reduced glutathione, a thiol, is essential to the survival of the cells of most aerobic organisms. It is present intracellularly and provides protection from hydroperoxides and free radicals. This unit describes a continuous spectrophotometric assay for reductase activity: it follows the reduction of glutathione disulfide to reduced glutathione by monitoring the oxidation of NADPH as visualized by a decrease in absorbance at 340 nm.
Assuntos
Ensaios Enzimáticos/métodos , Glutationa Redutase/análise , Animais , Citosol/enzimologia , Glutationa/metabolismo , HumanosAssuntos
Enzimas/metabolismo , Glutationa Transferase/metabolismo , Biblioteca de Peptídeos , Sítios de Ligação , Catálise , Enzimas/química , Enzimas/isolamento & purificação , Variação Genética , Glutationa Transferase/química , Glutationa Transferase/isolamento & purificação , Humanos , Indicadores e Reagentes , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoAssuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Sequência de Aminoácidos , Clonagem Molecular/métodos , Desoxirribonuclease I , Escherichia coli/genética , Biblioteca Gênica , Glutationa Transferase/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição/métodos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Moldes GenéticosRESUMO
Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to catalysis. The mutation had a marginal effect on the k(cat) value determined using S-D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K(m) value than the wild-type enzyme. The competitive inhibitor S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione had a 30-fold higher K(i) value towards the mutant, than that of the wild-type. Pre-equilibrium fluorescence studies with the inhibitor showed that this was due to a significantly increased off-rate for the mutant enzyme. The phenolic hydroxyl group of tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that this interaction makes a significant contribution to the binding of the active-site ligand.
Assuntos
Glutationa/metabolismo , Tioléster Hidrolases/metabolismo , Tirosina/metabolismo , Sítios de Ligação , Catálise , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Fenilalanina/química , Espectrometria de Fluorescência , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/química , Tirosina/químicaRESUMO
An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.
Assuntos
Glutationa Transferase/química , Mutação/genética , Substituição de Aminoácidos/genética , Cristalografia por Raios X , Estabilidade Enzimática , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.
Assuntos
Proteínas de Bactérias , Sequência Consenso/genética , Cianobactérias/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Biblioteca Gênica , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Composição de Bases , Sequência de Bases , Sítios de Ligação , Clonagem Molecular/métodos , Sequência Conservada/genética , DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Mutação/genética , Análise de Sequência de DNA , TermodinâmicaRESUMO
Human glutathione transferase M2-2 prevents the formation of neurotoxic aminochrome and dopachrome by catalyzing the conjugation of dopamine and dopa o-quinone with glutathione. NMR analysis of dopamine and dopa o-quinone-glutathione conjugates revealed that the addition of glutathione was at C-5 to form 5-S-glutathionyl-dopamine and 5-S-glutathionyl-dopa, respectively. Both conjugates were found to be resistant to oxidation by biological oxidizing agents such as O(2), H(2)O(2), and O(*-)(2), and the glutathione transferase-catalyzed reaction can therefore serve a neuroprotective antioxidant function.
Assuntos
Benzoquinonas/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Dopamina/metabolismo , Glutationa Transferase/metabolismo , Indolquinonas , Benzoquinonas/química , Cisteinildopa/análogos & derivados , Cisteinildopa/metabolismo , Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/metabolismo , Dopamina/química , Glutationa/metabolismo , Glutationa Transferase/química , Humanos , Indóis/metabolismo , Isoenzimas , Espectroscopia de Ressonância Magnética , Modelos Químicos , Oxirredução , Ligação Proteica , Quinonas/metabolismo , Fatores de TempoRESUMO
The evolution of proteins for novel functions involves point mutations and recombinations of domains or structural segments. Mimicking this process by rational design in vitro is still a major challenge. The present report demonstrates that the active site of the enzyme glutathione transferase (GST) A1-1 can be tailored for high catalytic efficiency with alkenals. The result is a >3,000-fold change in substrate selectivity involving a noteworthy change in preferred catalyzed reaction from aromatic nucleophilic substitution to Michael addition. The hydrophobic substrate binding pocket of GST A1-1 is formed by three structural modules, which were redesigned sequentially with four point mutations and the exchange of a helical segment. The substitutions were made to mimic first-sphere interactions with a substrate in GST A4-4, which naturally has high activity with alkenals. These substrates are toxic lipid peroxidation products of pathophysiological significance, and glutathione conjugation is a route of their inactivation. The final product of the sequential redesign of GST A1-1, mutant GIMFhelix, had a 300-fold increase in catalytic efficiency with nonenal and a >10 times decreased activity with 1-chloro-2,4-dinitrobenzene. In absolute values, GIMFhelix is more efficient than wild-type GST A4-4 with some alkenal substrates, with a k(cat)/K(m) value of 1.5 +/- 0. 1 10(6) M(-1) small middle dots(-1) for nonenal. The pKa value of the active-site Tyr-9 of GIMFhelix is 7.3 +/- 0.1, approaching the unusually low value of GST A4-4. Thus, rational redesign of the active-site region of an enzyme may be sufficient for the generation of efficient catalysts with altered chemical mechanism and novel selectivity.