Literature summary for 2.5.1.18 extracted from

Wongsantichon, J.; Robinson, R.C.; Ketterman, A.J.
Structural contributions of delta class glutathione transferase active site residues to catalysis (2010), Biochem. J., 428, 25-32.

Cloned(Commentary)

Cloned (Comment)	Organism
subcloning in Escherichia coli strain DH5alpha, expression in Escherichia coli strain BL21(DE3)	Anopheles dirus

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

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

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 [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

Organism	UniProt	Comment	Textmining
Anopheles dirus	Q9GN60	isozyme adGSTD4-4	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography to homogeneity	Anopheles dirus

Reaction

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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
delta class glutathione transferase	-	Anopheles dirus
GST	-	Anopheles dirus
GSTD4-4	-	Anopheles dirus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Anopheles dirus