Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase.

Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein-protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10(-7) M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex.

Glu-108 is essential for subunit assembly and dimer stability of D-phosphoglycerate dehydrogenase from Entamoeba histolytica.

D-phosphoglycerate dehydrogenase catalyses the first step of phosphorylated serine biosynthesis pathway by oxidizing 3-phosphoglycerate, a glycolysis intermediate into phosphohydroxsy pyruvate. For Entamoeba histolytica this pathway is an integral part of the cysteine metabolism, which is considered to be vital for growth and survival of the parasite. Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a homodimer at pH 7. Mild acidic conditions induce significant changes in the functional and structural features of the protein as observed by enzymatic activity, spectropolarimetric measurements and fluorescence spectroscopy. Most interestingly the oligomeric status of the protein was lost and a functionally inactive monomer was stabilized at pH 5. Computational modeling and molecular dynamic simulations show that dimeric assembly of EhPGDH was stabilized with the help of several inter-subunit non-covalent interactions and subunit dissociation at pH 5 can be attributed to protonation of acidic amino acid residues present at the dimer interface. Site directed mutagenesis studies suggest that Glu-108 is essential for subunit assembly as the E108A mutant existed as monomer even at pH 7. The studies unequivocally show that the electrostatic interactions at the dimer interface play a crucial role in the stability of the protein and a complete dimer is essentially required for optimal enzymatic activity.

Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite.

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol(-1) was observed between WT-protein and W101A mutant.

Comparative analysis of malate synthase G from Mycobacterium tuberculosis and E. coli: role of ionic interaction in modulation of structural and functional properties.

Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persistence through the upregulation of glyoxylate shunt. The malate synthase (MS), an important enzyme of the shunt belongs to the G isoform and expressed predominantly as monomer. Here we did a comparative unfolding studies of two homologous MS from Mycobacterium tuberculosis (MtbMS) and Escherichia coli (ecMS) using various biophysical techniques. Despite having high sequence identities, they show different structural, stability and functional properties. The study suggests that the differences in the stability and unfolding of the two enzymes are by virtue of differential electrostatic modulation unique to their respective molecular assembly.

Maximizing the native concentration and shelf life of protein: a multiobjective optimization to reduce aggregation.

A multiobjective optimization was performed to maximize native protein concentration and shelf life of ASD, using artificial neural network (ANN) and genetic algorithm (GA). Optimum pH, storage temperature, concentration of protein, and protein stabilizers (Glycerol, NaCl) were determined satisfying the twin objective: maximum relative area of the dimer peak (native state) after 48 h of storage, and maximum shelf life. The relative area of the dimer peak, obtained from size exclusion chromatography performed as per the central composite design (CCD), and shelf life (obtained as turbidity change) served as training targets for the ANN. The ANN was used to establish mathematical relationship between the inputs and targets (from CCD). GA was then used to optimize the above determinants of aggregation, maximizing the twin objectives of the network. An almost fourfold increase in shelf life (~196 h) was observed at the GA-predicted optimum (protein concentration: 6.49 mg/ml, storage temperature: 20.8 °C, Glycerol: 10.02%, NaCl: 51.65 mM and pH: 8.2). Since no aggregation was observed at the optimum till 48 h, all the protein was found at the dimer position with maximum relative area (64.49). Predictions of the finally adapted network also reveal that storage temperature and solvent glycerol concentration plays key role in deciding the degree of ASD aggregation. This multiobjective optimization strategy was also successfully applied in minimizing the batch culture period and determining optimum combination of medium components required for most economical production of actinomycin D.

Biophysical characterization of Entamoeba histolytica phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly.

We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T(m) and C(m) values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.

A functionally active dimer of mycobacterium tuberculosis malate synthase G.

Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The pleotropic function of this protein by virtue of its intracellular/extracellular localization and its behavior as an adhesin and virulence factor is quite enigmatic. Despite its importance in mycobacterium persistence, we do not know much about its biophysical and biochemical properties. Earlier reports suggest that the enzyme exists only as a monomer in prokaryotes; however, we observed the existence of both active monomer and dimer forms of the enzyme under physiological conditions. The dimeric form of the enzymes is more stable as compared to the monomeric form as evident from various biophysical parameters. In addition, the dimeric enzyme also shows enhanced stability against proteolysis than the monomers. Based on these studies, it seems that dimerization is an important factor in regulating stability. The differential localization and diverse functions of malate synthase other than its enzymatic role might be triggering the stabilization of the enzyme dimer and modulation of activity and stability in vivo.

Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship.

BACKGROUND: Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme. FINDINGS: EhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed. CONCLUSIONS: The study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.

Alkaline pH-dependent differential unfolding characteristics of mesophilic and thermophilic homologs of dimeric serine hydroxymethyltransferase.

Environmental variables such as pH can significantly influence the folding and stability of a protein molecule. In the present investigation, we compared the alkaline pH-induced unfolding of two homologous serine hydroxymethyltransferase from mesophilic Bacillus subtilis (bsSHMT) and thermophilic Bacillus stearothermophilus (bstSHMT) using various biophysical techniques. The thermophilic enzyme bstSHMT was found to be more resistant to alkaline denaturation compared to its mesophilic counterpart, bsSHMT. Unfolding studies using domain-swapped chimera, constructed by swapping the C-terminal domain of these two wild-type proteins, revealed that C-terminal domain plays a pivotal role in the folding, stability and subunit interaction of these proteins. Primary amino acid sequence analysis of the proteins showed that bsSHMT has six unconserved lysine residues in C-terminal domain, which are absent in bstSHMT. Chemical modification of lysine side chains resulted in stabilization of monomers, only in case of bsSHMT. Moreover, comparison between homology model of bsSHMT with the crystal structure of bstSHMT revealed that a small stretch of 11 amino acids at the end of C-terminal domain was found protruding outside the molecule as a flexible coiled structure in bsSHMT. Taken together these findings suggest that possibly the presence of these non-identical lysine moieties and a small extension of C-terminal domain may be responsible for low stability of bsSHMT under alkaline pH condition.

Conformational stability and energetics of Plasmodium falciparum glutaredoxin.

Glutaredoxins (Grxs), redox-active proteins with a typical -CPYC motif at their active sites, are involved in redox-regulatory processes and antioxidant defenses. The human malarial parasite Plasmodium falciparum possess a classical glutaredoxin (PfGrx) as well as a number of Grx-like proteins. In the present study, we investigated the unfolding energetics and conformational stability of PfGrx, using isothermal guanidine hydrochloride-induced and pH-dependent thermal denaturation. Reversible unfolding can be modeled using a two-state transition between the native and unfolded states. The structural topology of the protein was stable over a wide pH range from 3.0 to 11.0. Although the protein was thermally stable, it exhibited a small free energy of 1.56 kcal mol(-1) at 25 degrees C. The thermostability of PfGrx reached its maximum at pH 8.0, with a T(m) of 76.2 degrees C and a DeltaH(m) of 119 kcal mol(-1). To elucidate the factors underlying the thermostability, a protein stability curve was generated. Maximum stability occurred at around 47 degrees C, where the DeltaG_H2O(D) value was 4.30 kcal mol(-1). The high structural stability over a broad pH range, together with the capacity to endure very high temperatures, supports the notion that Grx can withstand a wide variety of conditions, allowing it to play a key role in cellular redox homeostasis. To the best of our knowledge, this work represents the first attempt to understand the energetic characteristics of a glutaredoxin in relation to accompanying structural changes.

Structural, functional and unfolding characteristics of glutathione S-transferase of Plasmodium vivax.

Glutathione S-transferases (GSTs) of Plasmodium parasites are potential targets for antimalarial drug and vaccine development. We investigated the equilibrium unfolding, functional activity regulation and stability characteristics of the unique GST of Plasmodium vivax (PvGST). Despite high sequence, structural, functional, and evolutionary similarity, the unfolding behavior of PvGST was significantly different from Plasmodium falciparum GST (PfGST). The unfolding pathway of PvGST was non-cooperative with stabilization of an inactive dimeric intermediate. The absence of any compact, folded monomeric intermediate during the unfolding transition suggests that inter-subunit interactions play an important role in stabilizing the protein. Presence of salts effectively inhibited PvGST enzymatic activity by quenching the nucleophilicity of the thiolate anion of GSH. Based on the present findings, together with our previous studies on PfGST, we propose that the regulation of GST enzymatic activity through a dimer-tetramer transition via GSH binding is an exclusive feature of Plasmodium.

Polysaccharide binding sites in hyaluronate lyase--crystal structures of native phage-encoded hyaluronate lyase and its complexes with ascorbic acid and lactose.

Hyaluronate lyases are a class of endoglycosaminidase enzymes with a high level of complexity and heterogeneity. The main function of the Streptococcus pyogenes bacteriophage protein hyaluronate lyase, HylP2, is to degrade hyaluronan into unsaturated disaccharide units. HylP2 was cloned, over-expressed and purified to homogeneity. The recombinant HylP2 exists as a homotrimer with a molecular mass of approximately 110 kDa under physiological conditions. The HylP2 was crystallized and the crystals were soaked in two separate reservoir solutions containing ascorbic acid and lactose, respectively. The crystal structures of native HylP2 and its two complexes with ascorbic acid and lactose have been determined. HylP2 folds into four distinct domains with a central core consisting of 16 antiparallel beta-strands forming an irregular triangular tube designated as triple-stranded beta-helix. The structures of complexes show that three molecules each of ascorbic acid and lactose bind to protein at the sugar binding groove in the triple-stranded beta-helix domain. Both ascorbic acid and lactose molecules occupy almost identical subsites in the long saccharide binding groove. Both ligands are involved in several hydrogen bonded interactions at each subsite. The binding characteristics and stereochemical properties indicate that Tyr264 may be involved in the catalytic activity of HylP2. The mutation of Tyr264 to Phe264 supports this observation.

Guanidine hydrochloride- and urea-induced unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase: stabilization of a functionally inactive holo-intermediate.

Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP+-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.

Evidence for inhibitory interaction of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) with Streptococcus pneumoniae hyaluronidase.

Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 microm. This is thus the first report of an endogenous protein inhibitor that may be used during natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1-mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research.

Self-assembly of bacteriophage-associated hyaluronate lyase (HYLP2) into an enzymatically active fibrillar film.

The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from pre-fibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular beta-sheet formation by the beta-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar film-like structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure.

Characterization of pyridoxal 5'- phosphate-binding domain and folding intermediate of Bacillus subtilis serine hydroxymethyltransferase: an autonomous folding domain.

The pyridoxal-5'-phosphate-binding domain (PLPbd) of bsSHMT (Bacillus subtilis serine hydroxymethyltransferase) was cloned and over-expressed in Escherichia coli. The recombinant protein was solublized, refolded and purified from inclusion bodies by rapid mixing followed by ion exchange chromatography. Structural and functional studies suggested the native form of the domain, which obtained as a monomer and had similar secondary and tertiary structural properties as when present in the bsSHMT. The domain also binds to the PLP however with slightly lesser affinity than the native enzyme. GdmCl (guanidium chloride)-induced equilibrium unfolding of the recombinant PLP-binding domain showed a single monophasic transition which corresponds with the second phase transition of the GdmCl-induced unfolding of bsSHMT. The results indicate that PLPbd of bsSHMT is an independent domain, which attains its tertiary structure before the dimerization of partially folded monomer and behaves as a single cooperative unfolding unit under equilibrium conditions.

Structural and stability characteristics of a monothiol glutaredoxin: glutaredoxin-like protein 1 from Plasmodium falciparum.

Recently discovered monothiol glutaredoxins with CXXS-active site sequence share a common structural motif and biochemical mechanism of action and are involved in multiple cellular functions. Here we report first studies on the structural and stability characterization of a monothiol glutaredoxin, in particular--PfGLP1. Our results demonstrate that in the native conformation, the enzyme has a compact core structure with a relatively flexible N-terminal portion having an open configuration. Comparative functional studies with the full-length and N-terminal truncated protein demonstrate that the flexible N-terminal portion does not play any significant role in functional activity of the protein. In contrast to other Grxs, PfGLP1 does not contain a Fe-S cluster. The pH dependent studies demonstrate that the protein is resistant to alkaline pH but highly sensitive to acidic pH and undergoes significant unfolding between pH 4 and 5. However, acidic conditions also do not induce complete unfolding of the enzyme. The protein is stabilized with a conformational free energy of about 3.2+/-0.1 kcal mol(-1). The protein is a highly cooperative molecule as during denaturant-induced equilibrium unfolding a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed.

Mycobacterium tuberculosis isocitrate lyase (MtbIcl): role of divalent cations in modulation of functional and structural properties.

Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg(2+) and Mn(2+) ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic alpha/beta barrel core domain of the enzyme and destabilize it. The inhibitors Zn(2+) and Cd(2+) interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl.

Toxoplasma gondii ferredoxin-NADP+ reductase: Role of ionic interactions in stabilization of native conformation and structural cooperativity.

The apicoplast and the proteins present therein are parasite-specific targets for chemotherapy of apicomplexan parasites. Ferredoxin-NADP(+) reductase (FNR) is an important enzyme present in the apicoplast of Toxoplasma gondii that operates as a general electron switch at the bifurcation step of many different electron transfer pathways. In spite of its importance as drug target not much structural information on the enzyme is available. Using fluorescence and CD spectroscopy in combination with enzyme activity measurement and size exclusion chromatography, we studied the pH-dependent changes in structural and functional properties and interdomain interactions in recombinant Toxoplasma gondii ferredoxin-NADP(+) reductase (TgFNR) to understand the interactions responsible for stabilization of native conformation and modulation of functional activity of the enzyme. Under physiological conditions, the recombinant TgFNR is stabilized in an open conformation. The open conformation of the enzyme was found to be essential for its optimum functioning, as induction of compactness/rigidity by modulation of pH, leads to decrease in the functional activity. In native conformation, strong interactions exist between the NADP(+)- and FAD-binding domains thus making the enzyme a structurally cooperative molecule. Under acidic conditions (pH about 4), the interdomain interactions present in native TgFNR were lost and the enzyme became structurally noncooperative. The pH-induced structural alterations in the NADP(+) binding domain, more precisely compaction of the conformation lead to its stabilization against thermal denaturation. The studies demonstrate the significance of electrostatic interactions both in stabilization of native conformation and maintenance of structural cooperativity in TgFNR.

Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase.

BACKGROUND: In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (approximately 98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out. RESULTS: Our data indicate that the dimer - and not the tetramer - is the active form of PfGST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of PfGST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the PfGST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it. CONCLUSION: This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of Plasmodium falciparum glutathione S-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers.