RESUMEN

INTRODUCTION: Bioequivalence testing for locally acting gastrointestinal drugs is a challenging issue for both regulatory authorities and pharmaceutical industries. The international regulatory framework has been characterized by the lack of specific bioequivalence tests that has generated a negative impact on the market competition and drug use in clinical practice. Areas covered: This review article provides an overview of the European Union and United States regulatory frameworks on bioequivalence criteria for locally acting gastrointestinal drugs, also discussing the most prominent scientific issues and advances that has been made in this field. A focus on oral modified release mesalamine formulations will be also provided, with practical examples of the regulatory pathways followed by pharmaceutical companies to determine bioequivalence. Expert commentary: The development of a scientific rationale to demonstrate bioequivalence in this field has been complex and often associated with uncertainties related to scientific and regulatory aspects. Only in recent years, thanks to advanced knowledge in this field, the criteria for bioequivalence assessment are undergoing substantial changes. This new scenario will likely result in a significant impact on pharmaceutical companies, promoting more competition through a clearer regulatory approach, conceived for streamlining the demonstration of therapeutic equivalence for locally acting gastrointestinal drugs.

Asunto(s)

Control de Medicamentos y Narcóticos , Fármacos Gastrointestinales/administración & dosificación , Mesalamina/administración & dosificación , Administración Oral , Preparaciones de Acción Retardada , Fármacos Gastrointestinales/farmacocinética , Humanos , Mesalamina/farmacocinética , Equivalencia Terapéutica

RESUMEN

Glutathione transferase reaches 0.5-0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S]≪[E]. As glutathione transferase lowers the pK(a) of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate-enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual "inverted" kinetics whereby the classical trends of v versus E and v versus S are reversed.

Asunto(s)

Biocatálisis , Glutatión Transferasa/metabolismo , Disulfuros/metabolismo , Glutatión/metabolismo , Humanos , Yodoacetatos , Isoenzimas/metabolismo , Cinética , Hígado/enzimología , Modelos Teóricos , Especificidad por Sustrato

RESUMEN

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC(50) of 2.3 +/- 0.6 micromol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC(50) of 4.5 +/- 0.9 micromol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH(2)-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/patología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glutatión Transferasa/antagonistas & inhibidores , Neoplasias Pulmonares/patología , Oxadiazoles/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma de Células Pequeñas/enzimología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Hexanoles/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/enzimología , Necrosis/inducido químicamente , Oxidación-Reducción/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Especificidad por Sustrato , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.

Asunto(s)

Glutatión Transferasa/metabolismo , Hepatocitos/enzimología , Membrana Nuclear/enzimología , Animales , Línea Celular Tumoral , Gutatión-S-Transferasa pi/metabolismo , Gutatión-S-Transferasa pi/fisiología , Glutatión Transferasa/fisiología , Humanos , Isoenzimas/metabolismo , Isoenzimas/fisiología , Masculino , Membrana Nuclear/química , Unión Proteica , Ratas , Ratas Wistar , Electricidad Estática

RESUMEN

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (GSTs) with extraordinary high affinity (K(D) = 10(-10) m). This complex is formed spontaneously through NO-mediated extraction of iron from ferritin and transferrin, in a reaction that requires only glutathione. In hepatocytes, DNDGIC may reach concentrations of 0.19 mm, apparently entirely bound to Alpha class GSTs, present in the cytosol at a concentration of about 0.3 mm. Surprisingly, about 20% of the dinitrosyl-glutathionyl-iron complex-GST is found to be associated with subcellular components, mainly the nucleus, as demonstrated in the accompanying paper (Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z., and Ricci, G. (2007) J. Biol. Chem. 282, 6372-6379). DNDGIC is a potent irreversible inhibitor of glutathione reductase, but the strong complex-GST interaction ensures full protection of glutathione reductase activity in the cells, and in vitro experiments show that damage to the reductase only occurs when the DNDGIC concentration exceeds the binding capacity of the intracellular GST pool. Because Pi class GSTs may exert a similar role in other cell types, we suggest that specific sequestering of DNDGIC by GSTs is a physiological protective mechanism operating in conditions of excessive levels of nitric oxide.

Asunto(s)

Glutatión Transferasa/fisiología , Hepatocitos/metabolismo , Hierro/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , Animales , Células Cultivadas , Gutatión-S-Transferasa pi/metabolismo , Glutatión Transferasa/metabolismo , Hepatocitos/citología , Humanos , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoprotein-positive tumors.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Apoptosis/efectos de los fármacos , Caspasas/fisiología , Inhibidores Enzimáticos/toxicidad , Glutatión Transferasa/antagonistas & inhibidores , Leucemia de Células T/enzimología , Leucemia de Células T/patología , Oxadiazoles/toxicidad , Piperazinas/toxicidad , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Enfermedad Aguda , Antineoplásicos/farmacología , Apoptosis/fisiología , Transporte Biológico/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Humanos , Cinética , Leucemia de Células T/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/fisiología , Oxadiazoles/síntesis química , Oxadiazoles/metabolismo , Fenotipo , Piperazinas/síntesis química , Piperazinas/metabolismo

RESUMEN

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione S-transferase (GST) P1-1, an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562 (human myeloid leukemia), HepG2 (human hepatic carcinoma), CCRF-CEM (human T-lymphoblastic leukemia), and GLC-4 (human small cell lung carcinoma). The LC50 values are in the micromolar/submicromolar range and are close to the IC50 values obtained with GSTP1-1, suggesting that the target of these molecules inside the cell is indeed this enzyme. The cytotoxic mechanism of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, the most effective GSTP1-1 inhibitor, has been carefully investigated in leukemic CCRF-CEM and K562 cell lines. Western blot and immunoprecipitation analyzes have shown that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol promotes in both cell lines the dissociation of the GSTP1-1 in a complex with c-jun NH2-terminal kinase (JNK). This process triggers a reactive oxygen species (ROS)-independent activation of the JNK-mediated pathway that results in a typical process of apoptosis. Besides this main pathway, in K562 cells, a ROS-mediated apoptosis partially occurs (about 30%) which involves the p38MAPK signal transduction pathway. The low concentration of this new compound needed to trigger cytotoxic effects on tumor cells and the low toxicity on mice indicate that the new 7-nitro-2,1,3-benzoxadiazole derivatives are promising anticancer agents.

Asunto(s)

Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Isoenzimas/antagonistas & inhibidores , Oxadiazoles/farmacología , Animales , Apoptosis/fisiología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Gutatión-S-Transferasa pi , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células K562 , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/enzimología , Leucemia de Células T/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Masculino , Ratones , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Asunto(s)

Glutatión Transferasa/química , Plasmodium falciparum/enzimología , 4-Cloro-7-nitrobenzofurazano/química , Animales , Asparagina/química , Sitios de Unión , Catálisis , Dimerización , Inhibidores Enzimáticos/farmacología , Evolución Molecular , Hemina/química , Concentración 50 Inhibidora , Cinética , Lisina , Modelos Químicos , Modelos Moleculares , Nitrógeno/química , Fosfatos/farmacología , Compuestos de Potasio/farmacología , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Electricidad Estática , Compuestos de Sulfhidrilo/química

RESUMEN

Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK.GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a sigma complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the sigma complex was the favored event. Docking studies demonstrated the likely localization of the sigma complex in the GST active sites and provide a structural explanation for its strong stabilization.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Oxadiazoles/química , Oxadiazoles/farmacología , Piperazinas/química , Piperazinas/farmacología , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Resistencia a Múltiples Medicamentos , Glutatión Transferasa/metabolismo , Hexanoles/química , Humanos , Células K562 , Cinética , Microscopía Fluorescente , Modelos Químicos , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia , Espectrofotometría , Compuestos de Sulfhidrilo/química , Temperatura , Factores de Tiempo

RESUMEN

The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions.

Asunto(s)

Glutatión Transferasa/metabolismo , Sitios de Unión , Catálisis , Ácido Dicloroacético/metabolismo , Escherichia coli/genética , Glutatión/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Análisis Espectral , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitroso-glutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione S-transferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1/2 = 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1/2 = 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.

Asunto(s)

Compuestos Ferrosos/metabolismo , Glutatión Transferasa/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Óxido Nítrico/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Retroalimentación Fisiológica , Femenino , Gutatión-S-Transferasa pi , Glutatión Transferasa/fisiología , Semivida , Humanos , Isoenzimas , Cinética , Hígado/metabolismo , Placenta/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley

RESUMEN

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (KD congruent with 10-10 m), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (KD congruent with 10-9 m). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (KD congruent with 10-7 m), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.

Asunto(s)

Glutatión Transferasa/metabolismo , Glutatión/análogos & derivados , Sitios de Unión , Evolución Molecular , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Glutatión/química , Glutatión/metabolismo , Gutatión-S-Transferasa pi , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiología , Estructura Cuaternaria de Proteína , Análisis Espectral

RESUMEN

Self-preservation is a typical property of living organisms, observed in the simplest prokaryotic cell as well as in the more complex pluricellular organisms. Surprisingly we found a self-preservation mechanism operating at the level of a single enzyme. Human glutathione transferase P1-1 operates in such a way towards either killer compounds (competitive and irreversible inhibitors) or physical factors (temperature and UV-rays), which could suppress its detoxicating and anti-cancer activity in the cell. This property, here termed 'co-operative self-preservation', is based on a structural intersubunit communication, by which one subunit, as a consequence of an inactivating modification, triggers a defence arrangement in the other subunit. Paradoxically this ability, developed during evolution for the survival of the cell, may not always be advantageous for us. In fact, glutathione transferase P1-1 is overexpressed in most tumour cells and pharmacological attempts to inhibit this enzyme in vivo, to prevent the drug resistance phenomenon during chemotherapy, may be thwarted by such self-preservation.

Asunto(s)

Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Sitios de Unión , Simulación por Computador , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Gutatión-S-Transferasa pi , Glutatión Transferasa/química , Glutatión Transferasa/efectos de los fármacos , Humanos , Isoenzimas/química , Isoenzimas/efectos de los fármacos , Modelos Moleculares , Subunidades de Proteína

RESUMEN

The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.

Asunto(s)

Evolución Molecular , Glutatión Transferasa/metabolismo , Oxidorreductasas , Proteus mirabilis/enzimología , Catálisis , Glutarredoxinas , Glutatión/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Proteínas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo