Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1

Self Cite

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK a ‫؍‬ 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k on and k off values at least hundred times lower (k on ‫؍‬ (2.7 ؎ 0.8) ؋ 10 4 M ؊1 s ؊1 , k off ‫؍‬ 36 ؎ 9 s ؊1 , at 37°C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK a value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Section: Discussionmentioning

confidence: 77%

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

Caccuri

Antonini

Board

et al. 2001

Self Cite

“…These results agree with findings on other GSTs, except for Theta class GSTs (38), where a highly conserved tyrosine, corresponding to Tyr 8 in PGDS, is thought to activate the SH group of the bound GSH by forming a hydrogen bond through the phenolic hydroxy group of the tyrosine (24,(32)(33)(34). In addition, it has been shown that the arginine residue corresponding to Arg 14 in rat PGDS is also involved in catalysis in some systems (35)(36)(37). Thus, we propose that the thiol group of GSH is activated by formation of a hydrogen bond with the phenolic hydroxyl of Tyr 8 and that Arg 14 assists the GSH activation by forming a salt bridge with the ␣-glutamyl carboxy anion of GSH to electrostatically neutralize the negative charge and to maintain the active configuration of the bound GSH (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Hematopoietic Prostaglandin D Synthase Activity Elucidated by Site-directed Mutagenesis

Pinzar¹,

Miyano²,

Kanaoka³

et al. 2000

Hematopoietic prostaglandin (PG) D synthase (PGDS)is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH 2 to PGD 2 and conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. We introduced site-directed mutations of 199, which are presumed to participate in catalysis or PGH 2 substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K m for glutathione and PGH 2 in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K m for glutathione was increased. Using 5,5-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. Prostaglandin (PG)1 D synthase (PGDS) is the key enzyme responsible for the formation of PGD 2 and its additional metabolites, the J series of PGs; and these prostanoids exhibit a variety of pharmacological activities and are involved in a number of physiological processes (1). For example, PGD 2 induces vasodilation and bronchoconstriction (2, 3); inhibits platelet aggregation (4); regulates body temperature (5, 6), hormone release (7), and nociception (8); and promotes sleep (9). Furthermore, ⌬ 12 -PGJ 2 inhibits the growth of various tumor cell lines (10), and 15-deoxy-⌬ 12,14 -PGJ 2 is an endogenous ligand for a nuclear receptor, peroxisome proliferator-activated receptor ␥ (11, 12).Two distinct types of PGDS have been isolated and characterized biochemically and immunologically; one is GSHdependent or hematopoietic PGDS (13,14), and the other is GSH-independent or lipocalin-type PGDS (15). Hematopoietic PGDS expressed in antigen-presenting, Kupffer, dendritic, Langerhans (16), megakaryoblastic (17), and mast (18, 19) cells of various organs is involved in the production of PGD 2 as an allergic and inflammatory mediator. In addition, it was characterized as a member of the glutathione S-transferase (GST) gene family (20), whose members are known to catalyze the conjugation of GSH to an electrophilic substrate (21).Recently, we cloned the cDNA for rat hematopoietic PGDS, crystallized the recombinant enzyme, and determined by x-ray crystallography the three-dimensional structure of the enzyme complexed with GSH (22). The deduced amino acid sequence and the tertiary structure of hematopoietic PGDS classified the enzyme as a member of the Sigma class of GSTs. The enzyme isomerizes PGH 2 to PGD 2 sele...

“…Clearly other components of the active site must contribute to the stabilization of GSH as a reactive thiolate ion. This has been shown to be the case in GSTA1-1 where Arg-15 contributes significantly to catalysis (59,60) in the presence or absence of Tyr-8, the primary active site residue.…”

Section: Gstomentioning

confidence: 92%

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

Menon

Board

2013

Self Cite

122

132

Background: Glutathionylation is a major post-translational modification that regulates protein function. Results: Human glutathione transferase Omega 1 (GSTO1-1) can catalyze the deglutathionylation of protein thiols in vitro and in cell culture. Conclusion: GSTO1-1, but not GSTO2-2, catalyzes the deglutathionylation of actin and other proteins under physiological conditions. Significance: Genetic variation in GSTO1-1-catalyzed deglutathionylation may influence the function of multiple proteins in signaling pathways.