The Isoenzymes of Glutathione Transferase

Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.

The glyoxalase system is present in the cytosol of cells and cellular organelles, particularly mitochondria. It is found throughout biological life and is thought to be ubiquitous. The widespread distribution and presence of the glyoxalase system in living organisms suggests it fulfils a function of fundamental importance to biological life. Recent investigations have brought new developments in the involvement of the glyoxalase in cell growth and vesicle mobilization, with increasing evidence of changes in the glyoxalase system during tumor growth and diabete mellitus, particularly relating to the development of associated clinical complications.

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

Structure and function of glutathione S-transferases

In Krebs-Ringer phosphate buffer, the rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate was first order with respect to the triose phosphate with rates constant values of 1.94 +/- 0.02 x 10(-5) s-1 (n = 18) and 1.54 +/- 0.02 x 10(-4) s-1 (n = 18) at 37 degrees C, respectively. The rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate in the presence of red blood cell lysate was not significantly different from the non-enzymatic value (P > 0.05). Methylglyoxal formation from glycerone phosphate was increased in the presence of triose phosphate isomerase but this may be due to the faster non-enzymatic formation from the glyceraldehyde 3-phosphate isomerisation product. For red blood cells in vitro, the predicted non-enzymatic rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate may account for the metabolic flux through the glyoxalase system. The reactivity of glycerone phosphate and glyceraldehyde 3-phosphate towards the non-enzymatic formation of methylglyoxal under physiological conditions suggests that methylglyoxal formation is unavoidable from the Embden-Meyerhof pathway.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17638.x

The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal.

Publisher Summary This chapter presents a procedure for the preparation of glyoxalase I that catalyzes the formation of S -2-hydroxyacylglutathione from a corresponding 2-oxoaldehyde and glutathione (GSH). A second enzyme, glyoxalase II, regenerates GSH by hydrolyzing the resulting thiolester. It is found that the formation of S -lactoylglutathione from methylglyoxal and GSH can be followed spectrophotometrically at 240 nm for analysis. Purification procedure are performed at about 5°, including the preparation of cytosol fraction; washing on sephadex G-25, carboxymethyl-cellulose, diethylaminoethyl cellulose column respectively; affinity chromatography with S-p -bromobenzylglutathione-sepharose 6B; washing on sephadex G-100; again affinity chromatography with S -p-bromobenzylglutathione- sepharose 6B; and chromatography on diethylaminoethyl-sepharose . The purified glyoxalase I is homogeneous in several analytical electrophoretic and chromatographic systems. In the presence of SDS, it dissociates into subunits of equal weight. Some of the known properties of glyoxalase I from the rat liver are presented in the chapter. Antibodies raised against the enzyme do not give precipitin lines with purified glyoxalase I from porcine erythrocytes, human erythrocytes, or Saccharomyces cerevisiae in Ouchterlony double-diffusion experiments.

[39] Glyoxalase I (rat liver)

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The Km value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn2+-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions.

Purification of glyoxalase I from human erythrocytes by the use of affinity chromatography and separation of the three isoenzymes

Publisher Summary Glyoxalase I catalyzes the formation of S-2-hydroxyacylglutathione from 2-oxoaldehydes, such as methylglyoxal and glutathione (GSH). This chapter describes the assay method, the purification procedure, and the properties of glyoxalase I isolated from human erythrocytes. Human glyoxalase I exists in the form of three isozymes that appear identical in their functional properties. The increase in absorbance at 240 nm because of thiolester formation from glutathione (GSH) and methylglyoxal is monitored spectrophotometrically. The purification procedure involves denaturation of hemoglobin, acetone fractionation, ammonium sulfate fractionation, affinity chromatography (I) on S-Hexylglutathione-Sepharose 6B, chromatography on Sephadex G-75, affinity chromatography II, chromatography on diethylaminoethyl (DEAE)-Sepharose, and separation of isozymes of glyoxalase I. The enzyme obtained by this procedure appears homogeneous as judged by various analytical procedures, except for the presence of the three isozymes. The enzyme is composed of two subunits of equal weight and contains one Zn per subunit that is essential to catalytic activity.

Gudrun Tibbelin

Papers

[39] Glyoxalase I (rat liver)

Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions.

Purification of glyoxalase I from human erythrocytes by the use of affinity chromatography and separation of the three isoenzymes

Glyoxalase I from human erythrocytes.

Crystallization of GST2, a human class alpha glutathione transferase