Enzyme Glyoxalase Research Articles

Kinetic analysis of the human erythrocyte glyoxalase system using 1H NMR and a computer model

1H NMR was used with methylglyoxal, purified by an HPLC technique, to study the kinetics of the human erythrocyte glyoxalase system. 1H NMR enabled the direct measurement of the time-dependent changes in concentrations of the two hydrates of methylglyoxal, which have not previously been directly measurable, as well as measurement of substrates and products of the glyoxalase enzyme system in the human red blood cell. A computer model of the reaction scheme was developed and NMR data numerically analyzed, thus allowing a complete kinetic description of the reactions. The rate constants describing the chemical equilibria between the hydrated species of methylglyoxal were determined by this numerical analysis or by a saturation-transfer technique, and found to be much slower (by several orders of magnitude) than previously determined by other methods. The kinetic parameters describing the enzyme-catalyzed reactions were also determined from experiments using a dilute haemolysate that was added to solutions of methylglyoxal and reduced glutathione (GSH). The maximal velocity of glyoxalase 1 is threefold greater (Vmax = 70.4 +/- 4.7 mmol.min-1.1 packed cells-1) than glyoxalase 2(Vmax = 24 +/- 5 mmol.min-1.1 packed cells-1) and it exhibits threefold-greater affinity for its substrate (Km = 0.46 +/- 0.04 mM) than the second enzyme (Km = 1.5 +/- 0.4 mM). Both enzymes are subject to competitive inhibition; glyoxalase 1 by reduced glutathione (KiGSH = 7.88 +/- 0.16 mM) and glyoxalase 2 by the hemithioacetal (HTA) of methylglyoxal and GSH (KiHTA = 0.29 +/- 0.04 mM).

Investigation on glyoxalase I inhibitors

Several classes of compounds — flavones, coumarins, S- and N-substituted glutathione analogs, transition state analogs, porphyrins, nucleotides and nucleosides — have been reported to inhibit the enzyme glyoxalase I. In the current study, examination of some of the aforementioned compounds has revealed that squaric acid does not function as an inhibitor of glyoxalase I and several other compounds are much less effective in this regard than previously reported. Several new potent inhibitors of yeast glyoxalase I have been identified. Compounds containing the tropolone structure were especially inhibitory. Glutathione adducts of benzoquinone and naphthoquinone were also inhibitory and may be of particular interest with regard to the toxicology of normal aromatic metabolites in vivo .

The human red blood cell glyoxalase system in diabetes mellitus

Methylglyoxal and other α-oxoaldehydes are formed from glycolytic intermediates and may be involved in the development of diabetic microangiopathy. Glyoxalase I and glyoxalase II metabolise methylglyoxal to d-lactic acid, via the intermediate S-d-lactoylglutathione. The activities of the glyoxalase enzymes and the concentrations of methylglyoxal and S-d-lactoylglutathione were measured in erythrocytes of 45 control and 85 diabetic subjects (41 with retinopathy and 44 uncomplicated). The concentration of S-d-lactoylglutathione was increased in diabetic patients vs. controls (21.4 ± 9.3 vs. 12.4 ± 4.8 μmol/l, P < 0.001), as was methylglyoxal (3.6 ± 2.3 vs. 1.4 ± 0.2 μmol/l, P < 0.001). There were no significant differences in the activities of glyoxalase I and glyoxalase II between diabetic patients and controls. For insulin-dependent patients only, those without retinopathy had a higher activity of glyoxalase II than those with retinopathy (P < 0.05). A group of age- and duration-matched insulin-dependent diabetic patients with retinopathy also had a higher activity of glyoxalase I compared with a group of diabetic patients without retinopathy (P < 0.025). This study provides evidence for elevated concentrations of oxoaldehydes in diabetes mellitus which might have pathogenic significance.

Theoretical studies of the electrostatic potential of some enzyme inhibitors using computer graphics techniques

A molecular graphics program is described for the depiction of electrostatic potentials on the van der Waals surface of molecules, using colored polygons. An example is given on the application of this method to the study of coumarins and flavones that inhibit the enzyme Glyoxalase I.

Modification of the glyoxalase system during the functional activation of human neutrophils

The glyoxalase system catalyses the metabolism of methylglyoxal to d-lactic acid, via the intermediate S- d- lactoylglutathione . It is present in human neutrophils and undergoes a significant modification during functional activation - induction of chemotaxis, phagocytosis and degranulation. During the activation of neutrophils with serum-opsonised zymosan and the tumour-promoting phorbol diester 12- O-tetrade-canoylphorbol 13-acetate, the activity of glyoxalase I increases and the activity of glyoxalase II decreases by 20–40% of their activities in resting cells, in the initial 10 min of the activation period. Determination of the Michaelis constant, K m, and the apparent maximum velocity, V max, for these enzymatic reactions indicates that the change in activity is due to a non-competitive activation and inhibition of glyoxalase I and glyoxalase II, respectively. This is consistent with a modification of the glyoxalase enzyme protein during the activation response. This modification occurs under aerobic and anaerobic incubation conditions. The concentration of S- d- lactoylglutathione increases approx. 100% of the resting cell concentration during the initial 10 min of the activation period. The presence of S- d- lactoylglutathione in neutrophils may be related to its ability to stimulate microtubule assembly.

Glyoxalase activity during differentiation of human leukaemia cells in vitro

The activities of glyoxalase I and glyoxalase II were determined in human promyelocytic leukaemia HL60 and erythroleukaemia K562 cells in culture. The activity of glyoxylase I is ca 10–20 times greater than the activity of glyoxalase II under assay conditions. When HL60 and K562 cell lines were incubated with N-methylformamide and 8-carbamoyl-3-methylimidazo-[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (CCRG 81045), substantial changes in the glyoxalase activities were induced. With HL60 cells, treatment with N-methylformamide and CCRG 81045, both of which induce functional differentiation of this cell line, there is a dose-dependent decrease in glyoxalase I activity and a concomitant dose-dependent increase in glyoxalase II activity, both of which are directly proportional to the number of differentiated cells. With K562 cells, N-methylformamide and CCRG 81045 induce an increase in both glyoxalase I and glyoxalase II activities, although only CCRG 81045 induces the appearance of haemoglobin producing cells. N-Methylformamide and CCRG 81045 do not activate or inhibit the activities of glyoxalase I and glyoxalase II from HL60 and K562 cells when studied in cell-free systems. The changes in the glyoxalase activities of HL60 and K562 cells during the incubations therefore appear to be due to alteration in the synthesis and/or regulatory modification of the glyoxalase enzymes induced by N-methylformamide and CCRG 81045. Despite the apparent disparity of the effect of differentiation on the glyoxalase system in the two cell lines, in both cases the glyoxalase I/glyoxalase II activity ratio decreases with the appearance of differentiated cells. Since glyoxalase II catalyses the rate-determining step in the glyoxalase system, this suggests that immature cells have an impaired capacity to metabolise S- d-lactoylglutathione.

The Simultaneous Separation of the Enzymes Glyoxalase I, Esterase D, and Phosphoglucomutase

A procedure for the multisystem analysis of bloodstains using the simultaneous separation of the enzymes glyoxalase I, esterase D, and phosphoglucomutase has been developed. The amount of bloodstain required has therefore been reduced threefold without any loss in resolution and sensitivity. Bloodstains at least seven weeks old have been correctly phenotyped in all three systems.

Nonresponsiveness to hepatitis B vaccine in health care workers. Results of revaccination and genetic typings.

Twenty-eight health care workers who had a poor antibody response when initially vaccinated with hepatitis B vaccine were revaccinated with three additional 20-microgram doses. Eight of the twenty nonresponders, who had levels of antibody to hepatitis B surface antigen (anti-HBs) of less than 8 estimated radioimmunoassay (RIA) units, and all 8 of the hyporesponders, who had anti-HBs levels of 8 or 16 RIA units, attained anti-HBs levels of 36 RIA units or more after revaccination. Tests for HLA-A, B, C, and DR; for complement proteins C2, C4A, C4B, and BF; and for the erythrocyte enzyme glyoxalase I were done in 17 nonresponders and 3 hyporesponders. Nine (45%) had HLA-DR7 and 8 (40%) had HLA-DR3, compared with an expected rate of 23% in the general population. At least one of two extended haplotypes (B44, DR7, FC31 or B8, DR3, SCO1) were detected in 6 of the 9 who did not respond to revaccination, compared with 2 of 11 who responded to a second course of vaccine. Poor responders to vaccine may benefit from revaccination, and genetic factors may modulate the immune response to vaccination.

ChemInform Abstract: S‐(NITROCARBOBENZOXY)GLUTATHIONES: POTENT COMPETITIVE INHIBITORS OF MAMMALIAN GLYOXALASE II

Three potent competitive inhibitors of mammalian liver glyoxalase II, the S-(o-, m-, and p-nitrocarbobenzoxy)-glutathiones, have been synthesized and studied. The Ki values of the ortho, meta, and para isomers, as inhibitors of rat liver glyoxalase II, were 15, 9, and 6.5 microM, respectively. While showing marked competitive inhibition of glyoxalase II, the glutathione derivatives were almost inactive as inhibitors of glyoxalase I. For example, with the para isomer, [I]0.5 values for rat liver glyoxalase I and II were 925 and 12 microM, respectively. This is in marked contrast to other glyoxalase II competitive inhibitors, which in general are even more effective against glyoxalase I. The S-(o-, m-, and p-nitrocarbobenzoxy)glutathiones have found utility as affinity ligands for the purification of rat liver glyoxalase II and may well have use in the study of the glyoxalase enzymes in vivo.

S-(nitrocarbobenzoxy)glutathiones: potent competitive inhibitors of mammalian glyoxalase II.

Three potent competitive inhibitors of mammalian liver glyoxalase II, the S-(o-, m-, and p-nitrocarbobenzoxy)-glutathiones, have been synthesized and studied. The Ki values of the ortho, meta, and para isomers, as inhibitors of rat liver glyoxalase II, were 15, 9, and 6.5 microM, respectively. While showing marked competitive inhibition of glyoxalase II, the glutathione derivatives were almost inactive as inhibitors of glyoxalase I. For example, with the para isomer, [I]0.5 values for rat liver glyoxalase I and II were 925 and 12 microM, respectively. This is in marked contrast to other glyoxalase II competitive inhibitors, which in general are even more effective against glyoxalase I. The S-(o-, m-, and p-nitrocarbobenzoxy)glutathiones have found utility as affinity ligands for the purification of rat liver glyoxalase II and may well have use in the study of the glyoxalase enzymes in vivo.

Model studies of the glyoxalase I reaction. Buffer-catalysed rearrangement to α-hydroxyacyl thiolesters of hemithioacetals from 2-mercaptoethanol with substituted arylglyoxals

A model system for the reaction catalysed by the enzyme glyoxalase I has been studied kinetically. A series of substituted arylglyoxals was synthesised. The hemithioacetals formed between these α-ketoaldehydes and β-mercaptoethanol in aqueous solutions at pH 9.2 underwent smooth rearrangement to the corresponding α-hydroxyacyl thiolester. The kinetics of this rearrangement were studied anaerobically and rate constants showed saturation dependence on thiol concentration {kobs=kmax.Kh[RSH]/(1 +Kh[RSH])} where kmax., refers to the limiting value of kobs at high thiol concentrations and Kh is the equilibrium constant for the hemithioacetal equilibrium. The rearrangement was catalysed by diazabicyclo[2.2.2]octane and second-order rate constants for this process followed a Hammett σ relationship with a ρ value of +0.90. Isotope effects in H2O- and D2O-based media for PhCOCHO and PhCOCDO, respectively, were measured at 30 °C to give kmax., (H/D)= 5.9 ± 0.9; Kh(H/D)= 0.24 ± 0.08. Activation parameters were obtained for isolated kmax. and Kh terms for phenylglyoxal with mercaptoethanol. The data obtained, along with literature information, allowed assignment of the rate-determining step to base-catalysed deprotonation at the C(1)–H site of the hemithioacetal (i.e. at the carbon α to the sulphur atom).

S-carbobenzoxyglutathione: a competitive inhibitor of mammalian glyoxalase II.

An effective competitive inhibitor of mammalian glyoxalase II has been synthesized and studied. The compound, S-carbobenzoxyglutathione, is almost totally inactive as an inhibitor of mammalian glyoxalase I. This is in marked contrast to other glyoxalase II competitive inhibitors, which in general are even more effective against glyoxalase I. S-Carbobenzoxyglutathione has found utility as an affinity ligand for the purification of rat liver glyoxalase II, and it may well have use in the study of the glyoxalase enzymes in vivo.

The glyoxalase system in rat blood.

AbstractThe 3-carbon keto-aldehyde, methylglyoxal (MeG), inhibits growth in bacterial and mammalian systems. The glyoxalase enzyme system catalyzes the catabolism of the α,β-dicarbonyl to form d-lactate with glutathione (GSH) as a cofactor. Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase, isomerizing; EC 4.4.1.5) and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) are present in mammalian tissues and have a high specific activity in red blood cells. In order to determine possible changes in the glyoxalase system with growth, the various components of the glyoxalase system were measured in the blood of male Sprague–Dawley rats aged 20 to 89 days. Glyoxalase I and II kinetic parameters were determined in potassium phosphate buffer at pH 6.8. Glutathione reductase activity, blood concentration of GSH and protein, plasma concentration of d-lactate, and hematocrit were also measured. The mean V for glyoxalase I was 81 μmole·mhr-1·ml-1 of red blood cells as compared to the glyoxalase II...

The tumor promoting phorbol diester, 12-0-tetradecanoylphorbol-13-acetate (TPA) increases glyoxalase I and decreases glyoxalase II activity in human polymorphonuclear leukocytes

Glyoxalase I and II catalyze the formation and breakdown of S-lactoylglutathione respectively. Recent studies have implicated this com-pound as a possible mediator of immune and inflammatory responses. Incubation of human polymorphonuclear leukocytes with the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate has been found to affect the activities of both glyoxalase enzymes in an interrelated manner. The diester either increases the activity of glyoxalase I or decreases the activity of glyoxalase II or has both effects. It is suggested that a subsequent increase in S-lactoylglutathione might mediate some or all of the effects of the phorbol diesters.

Mouse mitochondrial superoxide dismutase locus is on chromosome 17

The hamster X mouse hybridoma cell line GCL28 carries only one copy of mouse chromosome 17 but expresses H-2 antigens controlled by the major histocompatibility complex of the mouse. The cell line and clones derived from it were subjected to treatment with H-2 specific antisera and complement and a series of H-2 antigen-negative clones was produced. Typing of the clones for the mouse enzyme glyoxalase 1, which is encoded by an H-2-linked gene, revealed that the loss of H-2 antigen expression was accompanied by the loss of chromosomes 17 in these clones. This suggestion was verified by karyotype analysis of selected clones. Typing of the clones and subclones for the mouse mitochondrial superoxide dismutase (SOD-2) indicated complete concordance between loss of chromosome 17 and loss of SOD-2 activity. This finding suggests that the locus controlling the expression of SOD-2 is located on chromosome 17. Since a similar locus in the human is linked to HLA, the human major histocompatibility complex, extensive homology must exist between the mouse and human MHC-bearing chromosomes.

Inhibitors of glyoxalase enzymes

Both glyoxalase enzymes from various sources are inhibited by nucleotides, nucleosides and a series of structurally related compounds in an apparently cooperative manner. In general, aromatic compounds, which contain heterocyclic nitrogen and/or have amino groups on the aromatic ring, are effective inhibitors of these enzymes. Since intracellular concentrations of some of the nucleotides are significantly higher than the concentrations needed to inhibit both enzymes completely, the inhibitions observed may have physiological significance.

Involvement of glutathione in the inhibition of sea urchin egg mitosis by phenyl glyoxal.

Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone.

Effects of S-lactoylglutathione and inhibitors of glyoxalase I on histamine release from human leukocytes.

THE enzyme responsible for the conversion of methylglyoxal to lactic acid by animal tissues was first named glyoxalase1,2. It was later shown that there are two glyoxalase enzymes3. The first (glyoxalase I). converts methylglyoxal and reduced glutathione to S-lactoylglutathione and the second (glyoxalase II). converts this compound to D-lactic acid, regenerating glutathione in the process. These enzymes are very widely distributed4 and as yet have no clearly defined function(s). I describe here the enhancement of anti-IgE-induced histamine release by S-lactoylglutathione and its inhibition by an inhibitor of and an alternative substrate for glyoxalase I. These two compounds should, of course, decrease the production of endogenous S-lactoylglutathione. These experiments were undertaken for two reasons. First, a study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro5 while the release of histamine from leukocytes is a secretory process thought to require an intact microtubule system6,7. Second, concanavalin A has been shown to cause histamine release from human leukocytes8,9 and has also been found to activate the two glyoxalase enzymes in lymphocytes and polymorphonuclear leukocytes10.

Concanavalin A Increases Glyoxalase Enzyme Activities in Polymorphonuclear Leukocytes and Lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.

Glyoxalase I enzyme studies. 4. General base catalyzed enediol proton transfer rearrangement of methyl- and phenylglyoxalglutathionylhemithiol acetal to S-lactoyl- and S-mandeloylglutathione followed by hydrolysis. A model for the glyoxalase enzyme system

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGlyoxalase I enzyme studies. 4. General base catalyzed enediol proton transfer rearrangement of methyl- and phenylglyoxalglutathionylhemithiol acetal to S-lactoyl- and S-mandeloylglutathione followed by hydrolysis. A model for the glyoxalase enzyme systemStan S. Hall, Arthur M. Doweyko, and Frank JordanCite this: J. Am. Chem. Soc. 1978, 100, 18, 5934–5939Publication Date (Print):August 1, 1978Publication History Published online1 May 2002Published inissue 1 August 1978https://doi.org/10.1021/ja00486a054RIGHTS & PERMISSIONSArticle Views206Altmetric-Citations53LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (753 KB) Get e-Alerts Get e-Alerts