Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: Close structural connectivity between glutathione conjugation activity and non-substrate ligand binding (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S N Ar reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain-domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.
Flexibility of Helix 2 in the Human Glutathione Transferase P1-1
Journal of Biological Chemistry, 1998
Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of ␣-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M.
2005
Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.
The Journal of biological chemistry, 1996
Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2,4-dinitrobenzene (may be related to the frequency of enzyme motions. The observed low, viscosity-independent k cat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2002
Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (a9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the a9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k cat and k cat / K m for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the a9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate D 5 -androstene-3,17dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k cat /K m . The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Glutathione transferase A1-1; C-terminus; Viscosity effect 0167-4838/02/$ -see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 3 8 ( 0 2 ) 0 0 3 6 2 -X $ PII of original article S0167-4838(02)00286-8.
Journal of Biological Chemistry, 1998
Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of ␣-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M.
Acta Crystallographica Section D Biological Crystallography, 2006
Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 Å resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.
Journal of Molecular Biology, 2005
The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic a-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1$ S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand-protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex.
Journal of Biological Chemistry, 2010
Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.