Purification and characterization of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from human liver

Uotila, Lasse

doi:10.1021/bi00744a025

Cited by 98 publications

(72 citation statements)

References 20 publications

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“…The mechanism proposed for the glyoxalase IIcatalysed reaction involves an active site histidine residue. (1984, 1985, 1987a,b), Talesa et al (1988Talesa et al ( , 1989, Uotila (1973Uotila ( , 1979 (Ball & Vander Jagt, 1981). This is in keeping with the nucleophilicity of the imidazole group and the reactivity of N-acylimidazoles towards hydrolysis (Hall et al, 1978;Jencks & Carriuolo, 1959) (Fig.…”

Section: Glyoxalase IIsupporting

confidence: 67%

“…With S-lactoyl [CH3CH(OH)CO-], S-glycolyl (HOCH2CO-), S-mandelyl [PhCH(OH)CO-], S-glyceroyl [HOCH2CH(OH)CO-] and S-acetoacetyl (CH3COCH2CO-) glutathiones, the relative kinetic activity with glyoxalase II is maintained during purification. Pure glyoxalase II is inactive with thioesters of CoA and thioglycollate, indicating a high specificity for the glutathione moiety (Uotila, 1973 This has suggested that glyoxalase II is a very efficient catalyst for hydrolysis of S-D-lactoylglutathione .…”

Section: Glyoxalase IImentioning

confidence: 99%

“…Chemical modification studies of glyoxalase II have indicated the presence of arginine, histidine and lysine residues in the active site of glyoxalase II (Ball & Vander Jagt, 1981;Principato et al, 1985;Uotila, 1973). The mechanism proposed for the glyoxalase IIcatalysed reaction involves an active site histidine residue.…”

Section: Glyoxalase IImentioning

confidence: 99%

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The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life

Thornalley

1990

Biochemical Journal

732

502

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Section: Glyoxalase IIsupporting

confidence: 67%

Section: Glyoxalase IImentioning

confidence: 99%

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The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life

Thornalley

1990

Biochemical Journal

732

502

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“…The GLX2-2 gene has been successfully cloned and overexpressed in Escherichia coli yielding protein concentrations as high as 100 M (18). Early biochemical studies suggested that glyoxalase II, unlike glyoxalase I, does not require bound metal ions for activity (13,15,17,19). However, through the analysis of recombinant GLX2-2, we demonstrated that glyoxalase II is a Zn(II)-requiring enzyme (18).…”

mentioning

confidence: 85%

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

Zang

Hollman

Crawford

et al. 2001

Journal of Biological Chemistry

127

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Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or antiprotozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.The glyoxalase system consists of two enzymes that convert cytotoxic 2-oxoaldehydes into hydroxy acids utilizing the cofactor glutathione. Under physiological conditions, glyoxalase I (lactoylglutathione lyase) catalyzes the formation of S-D-lactoylglutathione (SLG) 1 in the presence of methylglyoxal and glutathione (1). Methylglyoxal, a cytotoxic compound that can inactivate proteins, modify guanylate residues in DNA, and create interstrand cross-links (2-4), is formed as a by-product of several biochemical reactions including that of triose-phosphate isomerase (2). Glyoxalase II (hydroxyacylglutathione hydrolase) hydrolyzes SLG to form D-lactic acid and regenerate glutathione (1). SLG is also known to exhibit cytotoxic effects through the inhibition of DNA synthesis (5). Therefore, the glyoxalase system is thought to play a major role in chemical detoxification (5, 6).The glyoxalase system has been the subject of intense study because of its potential implications in anti-protozoal and antitumor drug design (6 -8). Increased cellular levels of methylglyoxal and glyoxalase activity are associated with tumor cells, the malaria parasite, and several complications associated with diabetes (6). Inhibitors of glyoxalase I and glyoxalase II have been designed as potential anti-tumor agents; however, all inhibitors tested to date exhibited problems that limited their therapeutic usefulness (6 -9). It should be noted that these inhibition studies were all conducted with little or no information on the active site structure or the kinetic mechanism of the enzyme. Therefore, it is clear that a more detailed understanding of the active site of glyoxalase II and its reaction mechanism is essential to the rational design an...

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“…The existence of glutathione S-formyl and succinyl thioesters in human liver has been demonstrated, but * Without mersalyl. no biochemical role for these substances has been proposed (27,28). The enzymatic formation of thioesters involves GS' formed by the homolytic cleavage of GSSG.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

Kun

Kirsten

Sharma

1977

Proc. Natl. Acad. Sci. U.S.A.

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A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. (9) was also explained by the stringent requirement of preincubation of mitochondria with fluorocitrate in the absence of millimolar concentrations of citrate (cf. 7, 8), allowing for the formation of two protein-fluorocitrate thioesters (8).The present report is concerned with the identification of a mitochondrial enzyme system that catalyzes the formation of the 1:1 adduct of glutathione and citric acid. The adduct was identified as the thioester of glutathione, and kinetic evidence was also obtained that the mitochondrial extract contained, in addition to the thioester-forming enzyme system, a hydrolase capable of degrading the thioester. The citryl-glutathioneforming enzyme system proved to be the most sensitive catalytic component of mitochondria towards the irreversible inhibitory action of (-)erythrofluorocitrate, suggesting that the molecular site of action of this highly toxic agent has been identified. METHODSLysosome-free mitochondria and mitoplasts (defined as the matrix surrounded by the inner membrane) were isolated from rat livers as described (12). An extract of mitoplasts was prepared by stirring (at 00) of a suspension of mitoplasts (30 mg of protein per ml) in 0.25 M sucrose/mannitol medium (12) containing 1 mg of Brij 56 (Sigma Chemicals) per 10 mg of mitoplast protein for 15 min. The resulting lysate was diluted 1:3 with the medium; after removal of large granules (unbroken mitoplasts) by centrifugation at 10,000 X g (10 min at 40), the supernatant (containing soluble extract of the inner membrane and soluble proteins of the matrix and inner membrane vesicles) was subjected to ultracentrifugation (145,000 X g for 1 hr at 20). The clear supernatant was concentrated by centrifugation (at 20) in Amicon CF-25 membrane cones at 100 X g to one-fourth of the original volume. Approximately 250 mg of protein extract was obtained by this technique from 500 mg (protein) of mitoplast starting material. The concentrated extract was used directly in most experiments, unless specified, whereas the sediment containing the inner membrane vesicles was rehomogenized in the suspending medium. Both fractions were stored at -15°until used. The enzymatic activity of the soluble extract decayed slowly at -150 (about half of the activity was lost in 2 weeks) but much more rapidly after repeated thawing and refreezing. The activity was completely recovered when the stored extract was preincubated for 5-10 min with 5 mM reduced glutathione (GSH), demonstrating the essential role Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; citryl-SG and malyl-SG, glutathione thioesters of citric and malic acids, respectively. 4942

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Purification and characterization of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from human liver

Cited by 98 publications

References 20 publications

The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life

The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate

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