Cloned (Comment) | Organism |
---|---|
subcloning in Escherichia coli strain DH5alpha, expression in Escherichia coli strain BL21(DE3) | Anopheles dirus |
Crystallization (Comment) | Organism |
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purified recombinant wild-type and mutant enzymes F123A and Y119E in complex with S-hexyl glutathione, hanging drop vapour diffusion method at 16°C and 23°C, mixing of 0.002 ml of 10 mg/ml protein in 50 mM Tris-Cl, pH 7.5, and 5 mM S-hexyl glutathione, with 0.002 ml of precipitant solution containing 0.1 M cacodylate, pH 6.6, 29-34% of PEG4000, and 0.13-0.16 M of sodium 32 acetate, equilibration against 0.5 ml reservoir, 1-2 days, X-ray diffraction structure determination and analysis at 2.6-3.0 A resolution, molecular replacement | Anopheles dirus |
Protein Variants | Comment | Organism |
---|---|---|
F123A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Anopheles dirus |
F123E | site-directed mutagenesis, the mutant shows a negative effect on enzyme catalysis through either catalytic rate or binding affinity influence, but increased stability compared to the wild-type enzyme | Anopheles dirus |
F123X | site-directed mutagenesis, the mutants show reduced activity compared to the wild-type enzyme | Anopheles dirus |
Y111A | site-directed mutagenesis, the mutant shows a decreased kcat, but unaltered Km, compared to the wild-type enzyme | Anopheles dirus |
Y111E | site-directed mutagenesis, the mutant shows an approximately 14fold decreased GSH binding affinity, 225fold decreased catalytic efficiency, and a 15fold decreased kcat, but increased stability, relative to the wild-type enzyme. The 1,2-dichloro-4-nitrobenzene binding affinity is also decreased approximately 2.6fold relative to the wild-type enzyme probably due to conformational rearrangement of the H-site | Anopheles dirus |
Y111F | site-directed mutagenesis, the mutant shows a decreased kcat, but unaltered Km, compared to the wild-type enzyme | Anopheles dirus |
Y111H | site-directed mutagenesis, the mutant shows a slightly different catalytic rate compared to the wild-type enzyme, but the enzyme displays 4fold lower binding affinity towards GSH, as well as positive cooperativity | Anopheles dirus |
Y111S | site-directed mutagenesis, the mutant shows a decreased kcat, but unaltered Km, compared to the wild-type enzyme | Anopheles dirus |
Y118A | site-directed mutagenesis, the mutant shows 6.8fold increased half-life compared to the wild-type enzyme | Anopheles dirus |
Y119A | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Anopheles dirus |
Y119E | site-directed mutagenesis, the mutant shows 2-3fold reduced substrate binding affinity, but increased stability, compared to the wild-type enzyme | Anopheles dirus |
Y119F | site-directed mutagenesis, the mutant shows 18fold increased activity compared to the wild-type enzyme with substrate 1,2-dichloro-4-nitrobenzene, and a 9fold greater substrate binding affinity compared to the wild-type enzyme | Anopheles dirus |
Y119H | site-directed mutagenesis, the mutant is similar to the wild-type enzyme | Anopheles dirus |
Y119S | site-directed mutagenesis, the mutant shows 2-3fold reduced substrate binding affinity compared to the wild-type enzyme | Anopheles dirus |
General Stability | Organism |
---|---|
the aromatic zipper motif contributes to modulating the protein structure, which impacts on protein stability and the topological arrangement of the active site | Anopheles dirus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
S-hexyl-glutathione | binding structure with wild-type and mutant Y119E and F123A enzymes, overview | Anopheles dirus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | positive cooperativity for GSH and 1-chloro-2,4-dinitrobenzene, respectively, steady-state kinetics of wild-type and mutant enzymes, overview | Anopheles dirus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Anopheles dirus | Q9GN60 | isozyme adGSTD4-4 | - |
Purification (Comment) | Organism |
---|---|
recombinant enzyme from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography to homogeneity | Anopheles dirus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
RX + glutathione = HX + R-S-glutathione | active site structure: Tyr111 indirectly stabilizes glutathione binding, Tyr119 modulates hydrophobic substrate binding, and Phe123 indirectly modulates catalysis. An aromatic zipper in the H-site contributing a network of aromatic pi-pi interactions. Several residues of the cluster directly interact with the hydrophobic substrate while others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain II | Anopheles dirus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
1,2-dichloro-4-nitrobenzene + glutathione | binding structure with wild-type and mutant Y119E and F123A enzymes, overview | Anopheles dirus | S-(2-chloro-4-nitro-phenyl)glutathione + HCl | - |
? | |
1-chloro-2,4-dinitrobenzene + glutathione | Tyr119 not only plays a role in hydrophobic substrate binding but also in packing of the active site pocket, which influences chemical interactions involved in enzyme catalysis | Anopheles dirus | S-(2,4-dinitrophenyl)-glutathione + HCl | - |
? | |
4-nitrobenzyl chloride + glutathione | - |
Anopheles dirus | S-(4-nitrobenzyl)glutathione + HCl | - |
? | |
4-nitrophenethyl bromide + glutathione | - |
Anopheles dirus | S-(4-nitrophenyl)glutathione + HBr | - |
? | |
ethacrynic acid + glutathione | - |
Anopheles dirus | ? | - |
? | |
additional information | positive cooperativity upon GSH binding indicating that the residue replacement affects the communication between subunits of the dimeric enzyme | Anopheles dirus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the aromatic zipper motif contributes to modulating the protein structure, which impacts on protein stability and the topological arrangement of the active site | Anopheles dirus |
Synonyms | Comment | Organism |
---|---|---|
delta class glutathione transferase | - |
Anopheles dirus |
GST | - |
Anopheles dirus |
GSTD4-4 | - |
Anopheles dirus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Anopheles dirus |