Isomerization of (R)- and (S)-glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site.

The ability of glyoxalase I to isomerize both diastereomeric thiohemiacetals formed between glutathione and alpha-ketoaldehydes has been probed with stereochemically "locked" substrate analogues. Both (R)- and (S)-glutathiolactaldehyde (5 and 5') were unambiguously synthesized by employing the Sharpless epoxidation procedure as a key step. In the presence of human erythrocyte glyoxalase I, high-field 1H NMR analysis reveals that the R and S isomers (approximately 20 mM) are both converted to glutathiohydroxyacetone at rates of 0.8 and 0.4 s-1, respectively. This reaction is characterized by a nonstereospecific proton abstraction followed by a partially shielded proton transfer to the si face of the cis-enediol intermediate. Glyoxalase I catalyzes the exchange of the pro-S proton of glutathiohydroxyacetone with solvent deuterium. Glutathiohydroxyacetone was found to be a good competitive inhibitor of the normal glyoxalase I reaction (KI = 1.46 mM), suggesting that the slow processing rate of these compounds with respect to the normal thiohemiacetals is not due to poor binding. The results are consistent with a nonstereospecific proton abstraction and a stereospecific reprotonation at contiguous substrate carbons.     

Nonstereospecific substrate usage by glyoxalase I

Glyoxalase I operates on a mixture of rapidly interconverting diasteriomeric thiohemiacetals, formed in a preequilibrium step between glutathione and alpha-ketoaldehyde. That both diasteriomers are directly used as substrates by the enzyme from yeast and from porcine erythrocytes is an outcome of a series of isotope-trapping experiments in which pulse solutions composed of the two diasteriomeric thiohemiacetals, due to [3H]glutathione and phenylglyoxal, are rapidly mixed with chase solutions containing excess unlabeled glutathione and successively increasing concentrations of glyoxalase I. As the enzyme approaches infinite concentration in the chase solution, the radioactivity incorporated into the S-mandeloylglutathione product approaches 100% of the total radioactivity due to both diasteriomers from the pulse solution. The special properties of the active site that allow the enzyme to accommodate both diasteriomeric substrate forms may also account for the fact that the cis and the trans isomers of various para-substituted S-(phenylethenyl)glutathione derivatives are both strong competitive inhibitors of the enzyme. A catalytic mechanism is proposed for glyoxalase I involving catalyzed interconversion of the bound diasteriomeric thiohemiacetals before transformation to final product.     

Inhibition of glyoxalase I by the enediol mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione. The possible basis of a tumor-selective anticancer strategy.

In principle, competitive inhibitors of glyoxalase I that also serve as substrates for the thioester hydrolase glyoxalase II might function as tumor-selective anti-cancer agents, given the role of these enzymes in removing cytotoxic methylglyoxal from cells and the observation that glyoxalase II activity is abnormally low in some types of cancer cells. In support of the feasibility of this anticancer strategy, an inhibitor of this type has been synthesized by a thioester-interchange reaction between glutathione and N-hydroxy-N-methylcarbamate 4-chlorophenyl ester to give S-(N-hydroxy-N-methylcarbamoyl)glutathione (1). This compound was designed to be a tight-binding inhibitor of glyoxalase I, on the basis of its stereoelectronic similarity to the enediol(ate) intermediate that forms along the reaction pathway of this enzyme. Indeed, 1 is a competitive inhibitor of yeast glyoxalase I, with an inhibition constant (Ki = 68 microM) that is approximately 30-fold lower than that reported for S-D-lactoylglutathione and approximately 7-fold lower than the Km for glutathione-methylglyoxal thiohemiacetal. In addition, 1 is a substrate for bovine liver glyoxalase II, with a Km (0.48 mM) approximately equal to that of the normal substrate S-D-lactoyglutathione and a kcat approximately 2 x 10(-5)-fold that of the normal substrate. Membrane transport studies show that 1 can be delivered into human erythrocytes (used here as a model cell) either by direct diffusion of 1 across the cell membrane or by more rapid diffusion of the glycylethyl ester of 1 across the cell membrane, followed by the catalyzed hydrolysis of the ester to give 1.     

Caution: the glycylmethyl and glycylethyl esters of glutathione are substrates for glyoxalase I.

The glycylmethyl and glycylethyl esters of glutathione have been synthesized and carefully characterized by both 1H-NMR and tandem FAB mass spectrometry. Contrary to previously published studies, these compounds (as their methylglyoxal-thiohemiacetals) do indeed serve as moderately efficient substrates for yeast glyoxalase I, with kcat values that are approx. 3-fold smaller and Km values that are approx. 3-fold larger than those of the thiohemiacetal formed from glutathione. Product inhibition studies show that the glycylmethyl and glycylethyl esters of (S)-D-lactoylglutathione bind approx. 1.4-fold less tightly to the active site than (S)-D-lactoylglutathione. These observations exclude an essential role for the glycyl-CO2- of substrate in active site binding and catalysis.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.     

The protease inhibitor chagasin of Trypanosoma cruzi adopts an immunoglobulin-type fold and may have arisen by horizontal gene transfer

Methylglyoxal metabolism was studied during Saccharomyces cerevisiae grown with D-glucose as the sole carbon and energy source. Using for the first time a specific assay for methylglyoxal in yeast, metabolic fluxes of its formation and D-lactate production were determined. D-Glucose consumption and ethanol production were determined during growth. Metabolic fluxes were also determined in situ, at the glycolytic triose phosphate levels and glyoxalase pathway. Maximum fluxes of ethanol production and glucose consumption correspond to maxima of methylglyoxal and D-lactate formation fluxes during growth. Methylglyoxal formation is quantitatively related to glycolysis, representing 0.3% of the total glycolytic flux in S. cerevisiae.     

The sterochemical configuration of the lactoyl group of S-lactoylglutathionine formed by the action of glyoxalase I from porcine erythrocytes and yeast

S-Lactoylglutatione formed by the reaction between methylglyoxal and glutathione, catalyzed by glyoxalase I, has been isolated by means of gel filtration. The product was analyzed for content of thiolester, thiol, and d- and l-lactate before and after hydrolysis of the thiolester linkage. From the results it is concluded that glyoxalase I from both porcine erythrocytes and yeast stereospecifically transfers hydrogen to form S-d-lactoylglutathione from methylglyoxal and glutathione.     

D-lactate metabolism in starved Octopus ocellatus

The concentrations of D- and L-lactate, methylglyoxal and pyruvate were measured in tissues of normal and starved Octopus ocellatus. D-Lactate was always more abundant than L-lactate in the tissues. D-Lactate, pyruvate and methylglyoxal were present in 320, 94 and 43 times higher concentrations in tentacle of O. ocellatus of control group than those in normal rat skeletal muscle. The D-lactate concentration in the tentacle of O. ocellatus was 17-fold higher than that in Octopus vulgars. The activities of enzymes involved with D-lactate metabolism such as pyruvate kinase, octopine dehydrogenase, glyoxalase I and II and lactate dehydrogenase were measured in those tissues. The activities of glyoxalase I and II, and D-lactate dehydrogenase were increased in mantle and tentacle of starved octopus, while the levels of D-lactate and related metabolites were lowered in these tissues. The experimental results presented in this report and up to the present indicate that D-lactate is actively used for energy production in the tentacle and mantle of the starved animals. In octopus, especially starved octopus D-lactate was actively produced from methylglyoxal, which is formed via aminoacetone from threonine and glycine.     

Cumene peroxide and Fe2+-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase

Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA ↔ methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 ± 8 μM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

The glyoxalase system of malaria parasites--implications for cell biology and general glyoxalase research

Malaria parasites of the genus Plasmodium have developed sophisticated mechanisms to benefit from the nutrient-rich environments of their hosts. For example, by hiding in red blood cells, they found a secure way to tap into the glucose supply of vertebrates. The high-power metabolism of Plasmodium leads not only to a significantly increased glucose consumption of infected erythrocytes, but also to an elevated production of D-lactate from methylglyoxal. The latter substance is a harmful by-product from glycolysis that is detoxified by the ubiquitous glyoxalase system. This system consists of reduced glutathione and two enzymes, the glyoxalases 1 and 2. Inhibition of the glyoxalases in the host/parasite unit is expected to be highly detrimental to the parasite. Moreover, by studying Plasmodium isozymes, physiological functions of the system beyond methylglyoxal conversion became prima facie obvious: (i) the two different active sites of glyoxalase 1 as well as the existence of (insular) glyoxalases in the apicoplast point to alternative substrates and metabolic pathways. (ii) The allostery of glyoxlase 1 and the monomer-dimer equilibrium of glyoxalase 2 suggest novel regulatory features of these enzymes. Here we review the current knowledge on the glyoxalase systems of the host/parasite unit, discuss their potential as drug target and summarize new hypotheses on glyoxalases with respect to general cell biology.     

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-tetradecanoylphorbol 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, Km, and the apparent maximum velocity, Vmax, 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.     

Kinetic competence of an externally generated dienol intermediate with steroid isomerase.

The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.     

A New Role of Phosphoglucose Isomerase. Involvement of the Glycolytic Enzyme in Aldehyde Metabolism

Lipid peroxidation in biological membranes is accompanied by malonic dialdehyde (MDA) formation, but the problem of its further metabolism in cytoplasm remains unsolved. The experimental data obtained in this work showed that the liver fraction prepared by centrifugation at 10,000g contained phosphoglucose isomerase and enzymes of the glyoxalase system. In this fraction in the presence of GSH there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes (MDA conversion to methylglyoxal and further to neutral D-lactate). This means that MDA is slowly accumulated because it is a substrate of aldehyde isomerase (MDA <--> methylglyoxal). Most probably, phosphoglucose isomerase serves as this enzyme. We concluded that D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation

The glyoxalase pathway of Leishmania infantum was kinetically characterized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A K(m) of 0.253 mm and a V of 0.21 micromol.min(-1).mg(-1)for glyoxalase I, and a K(m) of 0.098 mm and a V of 0.18 micromol.min(-1).mg(-1) for glyoxalase II, were obtained. Modelling and computer simulation were used for evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in Leishmania infantum. A sensitivity analysis of the pathway was performed using experimentally validated kinetic models and experimentally determined metabolite concentrations and kinetic parameters. The measurement of metabolites in L. infantum involved the identification and quantification of methylglyoxal and intracellular thiols. Methylglyoxal formation in L. infantum is nonenzymatic. The sensitivity analysis revealed that the most critical parameters for controlling the intracellular concentration of methylglyoxal are its formation rate and the concentration of trypanothione. Glyoxalase I and II activities play only a minor role in maintaining a low intracellular methylglyoxal concentration. The importance of the glyoxalase pathway as a therapeutic target is very small, compared to the much greater effects caused by decreasing trypanothione concentration or increasing methylglyoxal concentration.     

Equilibrium constants for the formation of glyoxylate thiohemiacetals and kinetic constants for their oxidation by O2 catalyzed by l-hydroxy acid oxidase

Glyoxylate thiohemiacetal formation constants (defined as the concentration of thiohemiacetal divided by the concentration of thiol and the total concentration of hydrated and unhydrated glyoxylate) were determined at 25°C and pH 7.4 for a variety of thiols using two independent methods, and were found to be in the range of 0.2 to 1.7 mm^?1. Under the same conditions the hydration constant for glyoxylate (defined as the concentration of the hydrate divided by the concentration of the free aldehyde) was determined to be 163 ± 7. This information is used in conjunction with kinetic data to calculate kinetic constants for the oxidation of the thiohemiacetals by O₂ catalyzed by rat kidney l-hydroxy acid oxidase. The results further indicate that several such thiohemiacetals are excellent substrates, and suggest that one or more of them may be the physiological reactant for this enzyme.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin

Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.