RESUMEN
Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs.
Asunto(s)
Ácido Ascórbico/química , Glutatión Transferasa/química , Glutatión Transferasa/ultraestructura , Pennisetum/metabolismo , Proteínas de Plantas/química , Sitios de Unión , Activación Enzimática , Ligandos , Simulación del Acoplamiento Molecular , Proteínas de Plantas/análisis , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu).
Asunto(s)
Glutatión Transferasa/genética , Glutatión Transferasa/ultraestructura , Modelos Moleculares , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Biología Computacional , Bases de Datos de Proteínas , Glutatión/química , Estructura Terciaria de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.
The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.
Asunto(s)
Glucosidasas/análisis , Glucosidasas/biosíntesis , Glutatión Transferasa/análisis , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/clasificación , Glutatión Transferasa/farmacología , Glutatión Transferasa/química , Glutatión Transferasa/síntesis química , Glutatión Transferasa/ultraestructura , Oligosacáridos/aislamiento & purificación , Oligosacáridos/análisis , Oligosacáridos/genética , Oligosacáridos/química , Oligosacáridos/síntesis química , Oligosacáridos/ultraestructura , OligosacáridosRESUMEN
Human leukotriene C(4) synthase (LTC(4)S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC(4)S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC(4)S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.
Asunto(s)
Cristalización/métodos , Glutatión Transferasa/química , Microscopía por Crioelectrón , Detergentes/química , Glutatión Transferasa/ultraestructura , Glicerol/química , Humanos , Sales (Química)/química , TemperaturaRESUMEN
Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Dicroismo Circular , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Glutatión Transferasa/ultraestructura , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Humanos , Técnicas In Vitro , Cinética , Luz , Microscopía Electrónica de Transmisión , Modelos Biológicos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Péptidos/genética , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Dispersión de Radiación , Solubilidad , Termodinámica , Expansión de Repetición de TrinucleótidoRESUMEN
Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.
Asunto(s)
Glutatión Transferasa/química , Proteínas de la Membrana/química , Microsomas Hepáticos/enzimología , Estrés Oxidativo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Etilmaleimida/química , Glutatión/química , Glutatión Transferasa/metabolismo , Glutatión Transferasa/ultraestructura , Cinética , Masculino , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteolípidos/química , Proteolípidos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.
Asunto(s)
Cristalinas/química , Glutatión Transferasa/química , Animales , Fenómenos Biofísicos , Biofisica , Biopolímeros/química , Cristalinas/ultraestructura , Glutatión Transferasa/ultraestructura , Guanidina , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Microscopía Electrónica , Modelos Moleculares , Octopodiformes/química , Octopodiformes/enzimología , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Cloruro de Sodio , Espectrometría de Fluorescencia , Electricidad Estática , Temperatura , UreaRESUMEN
A technique for detecting the displacement of micron-sized optically trapped probes using far-field interference is introduced, theoretically explained, and used to study the motility of the ncd motor protein. Bead motions in the focal plane relative to the optical trap were detected by measuring laser intensity shifts in the back-focal plane of the microscope condenser by projection on a quadrant diode. This detection method is two-dimensional, largely independent of the position of the trap in the field of view and has approximately 10-micros time resolution. The high resolution makes it possible to apply spectral analysis to measure dynamic parameters such as local viscosity and attachment compliance. A simple quantitative theory for back-focal-plane detection was derived that shows that the laser intensity shifts are caused primarily by a far-field interference effect. The theory predicts the detector response to bead displacement, without adjustable parameters, with good accuracy. To demonstrate the potential of the method, the ATP-dependent motility of ncd, a kinesin-related motor protein, was observed with an in vitro bead assay. A fusion protein consisting of truncated ncd (amino acids 195-685) fused with glutathione-S-transferase was adsorbed to silica beads, and the axial and lateral motions of the beads along the microtubule surface were observed with high spatial and temporal resolution. The average axial velocity of the ncd-coated beads was 230 +/- 30 nm/s (average +/- SD). Spectral analysis of bead motion showed the increase in viscous drag near the surface; we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the nonhydrolyzable nucleotide adenylylimidodiphosphate.
Asunto(s)
Proteínas de Drosophila , Cinesinas/química , Cinesinas/ultraestructura , Microscopía de Interferencia/instrumentación , Microscopía de Interferencia/métodos , Fragmentos de Péptidos/química , Calibración , Clonación de Organismos , Diseño de Equipo , Escherichia coli , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/química , Glutatión Transferasa/ultraestructura , Cinesinas/biosíntesis , Cinética , Rayos Láser , Microtúbulos/ultraestructura , Modelos Químicos , Fragmentos de Péptidos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/ultraestructura , Sensibilidad y Especificidad , Factores de Tiempo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , ViscosidadRESUMEN
The crystal structure of the sigma class glutathione transferase from squid digestive gland in complex with S-(3-iodobenzyl)glutathione reveals a third binding site for the glutathione conjugate besides the two in the active sites of the dimer. The additional binding site is near the crystallographic two-fold axis between the two alpha 4-turn-alpha 5 motifs. The principal binding interactions with the conjugate include specific electrostatic interactions between the peptide and the two subunits and a hydrophobic cavity found across the two-fold axis that accommodates the 3-iodobenzyl group. Thus, two identical, symmetry-related but mutually exclusive binding modes for the third conjugate are observed. The hydrophobic pocket is about 14 A from the hydroxyl group of Tyr-7 in the active site. This site is a potential transport binding site for hydrophobic molecules or their glutathione conjugates.
Asunto(s)
Glutatión Transferasa/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Decapodiformes/enzimología , Glutatión Transferasa/ultraestructura , Secuencias Hélice-Giro-Hélice , Sustancias Macromoleculares , Modelos Moleculares , Proteínas RecombinantesRESUMEN
Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.
Asunto(s)
Dípteros/enzimología , Glutatión Transferasa/ultraestructura , Animales , Cristalografía por Rayos XRESUMEN
Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pKa) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases.
Asunto(s)
Glutatión Transferasa/ultraestructura , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Citosol/enzimología , Glutatión/química , Glutatión Transferasa/química , Ligandos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , PorcinosRESUMEN
The three-dimensional structures of a class mu glutathione transferase in complex with a transition-state analogue, 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate, and a product, 1-(S-glutathionyl)-2,4-dinitrobenzene, of a nucleophilic aromatic substitution (SNAr) reaction have been determined at 1.9- and 2.0-A resolution, respectively. The two structures represent snapshots along the reaction coordinate for the enzyme-catalyzed reaction of glutathione with 1-chloro-2,4-dinitrobenzene and reveal specific interactions between the enzyme, intermediate, and product that are important in catalysis. The geometries of the intermediate and product are used to postulate reaction coordinate motion during catalysis.
Asunto(s)
Glutatión Transferasa/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Glutatión Transferasa/ultraestructura , Enlace de Hidrógeno , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.
Asunto(s)
Glutatión Transferasa/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía , Glutatión/química , Glutatión Transferasa/clasificación , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes , Alineación de Secuencia , Programas Informáticos , Difracción de Rayos XRESUMEN
The present study is aimed to elucidate the changes in glutathione S-transferase (GST) activity and GST subunit components in primary cultured rat hepatocytes. Enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as cosubstrate. The activity decreased at 48 hr, and subsequently increased and returned to levels initially observed at 12 hr by 120 hr. Phenobarbital caused an induction of GST activity in culture at 72 and 168 hr. Immunocytochemical studies were performed using a peroxidase-anti-peroxidase technique with three polyclonal antibodies: anti-Ya, Yb1 and Yp. With anti-Ya, hepatocytes were persistently positive up to 144 hr in cell culture. With anti-Yb1, hepatocytes were positive at 24 hr, though positivity then gradually decreased. On the other hand, with anti-Yp, cells were almost negative at 48 hr and became obviously positive at 96 hr. Immunoelectron microscopy with anti-Yb1 using the avidin-biotin ferritin method revealed ferritin particles in the ribosomes on endoplasmic reticulum as well as in the free cytoplasmic space. In conclusion, the GST subunit components are in a state of dynamic change in cultured rat hepatocytes, and overall time-dependent increase in the total activity of the enzyme can be accounted for by increased expression of the Yp subunit. Finally, the intracellular localization of Yb1 subunit was clarified in the present report.
Asunto(s)
Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Hígado/enzimología , Animales , Células Cultivadas , Glutatión Transferasa/ultraestructura , Inmunohistoquímica , Hígado/ultraestructura , Masculino , Fenobarbital/farmacología , RatasRESUMEN
The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.
Asunto(s)
Glutatión Transferasa/ultraestructura , Glutatión/análogos & derivados , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía , Análisis de Fourier , Glutatión/metabolismo , Glutatión Transferasa/antagonistas & inhibidores , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Aceleradores de Partículas , Placenta/enzimología , Unión Proteica , Conformación Proteica , PorcinosRESUMEN
Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.