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.     

Functional residues on the enzyme active site of glyoxalase I from bovine brain

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.     

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.     

A reinvestigation of inhibitors of glyoxalase I

It has been reported earlier that nucleotides, nucleosides and a series of structurally related compounds as well as compounds based on transition state analogy inhibit yeast glyoxalase I. In our study on the metabolic regulation of glyoxalase I, we have found that nucleotides such as ATP, GTP and different classes of other reagents based on transition state analogy (D-isoascorbate, dihydroxyfumaric acid, rhodizonic acid) do not inhibit yeast or goat liver glyoxalase I. The reported inhibition of glyoxalase I by these compounds has been found to be due to the interference of these compounds with the absorbancy at 240 nm of S-D-lactoylglutathione formed by the glyoxalase I reaction. Glyoxalase I from goat liver has been found to be strongly and competitively inhibited by lactaldehyde. But, lactaldehyde has very little inhibitory effect on yeast glyoxalase I. Lactaldehyde is formed from methylglyoxal, the substrate for glyoxalase I by the enzyme methylglyoxal reductase. D-Lactaldehyde inhibits the liver enzyme more strongly than L-lactaldehyde.     

Yeast glyoxalase I is a monomeric enzyme with two active sites

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.     

Electron paramagnetic resonance study of the active site of copper-substituted human glyoxalase I

Zn2+ in native glyoxalase I from human erythrocytes can be replaced by Cu2+, giving an inactive enzyme. Cu2+ was demonstrated to compete with the activating metals Zn2+ and Mn2+, indicating a common binding site on the enzyme for these metal ions. The electron paramagnetic resonance (EPR) spectra of 63Cu(II) glyoxalase I at 77 K and of its complexes with glutathione and some glutathione derivatives are characteristic of Cu2+ in an elongated octahedral coordination (g parallel = 2.34, g perpendicular = 2.09, and A parallel = 14.2 mT). The low-field bands of the free enzyme are asymmetric and become symmetrical upon addition of glutathione or S-(p-bromobenzyl)glutathione but not S-(D-lactoyl)glutathione. The results indicate the existence of two conformations of Cu(II) glyoxalase I, in agreement with the effects caused by these compounds on the protein fluorescence. The copper hyperfine line at low field in the EPR spectrum of the S-(p-bromobenzyl)glutathione complex of 63Cu(II) glyoxalase I shows a triplet structure, indicative of coupling to one nitrogen ligand in the equatorial plane. Similar results were obtained with the glutathione complex. By addition of the spectrum of the S-(p-bromobenzyl)glutathione complex and a spectrum corresponding to two nitrogen ligands with two different coupling constants, a good fit was obtained for the low-field region of the asymmetric spectrum of free 63Cu(II) glyoxalase I. The first two spectra are assumed to correspond to two separate conformational states of the enzyme. The results demonstrate that at least one nitrogen ligand is involved in the binding of Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Sexual response of Saccharomyces cerevisiae: phosphorylation of yeast glyoxalase I by a cell extract of mating factor-treated cells

The phosphorylation of glyoxalase I was observed when the phosphatase-treated enzyme was incubated in the presence of [gamma-32P]ATP and a cell extract prepared from alpha-type yeast cells which had been treated with the culture supernatant of a-type yeast cells. The phosphorylated protein was identified as glyoxalase I by using anti-glyoxalase I rabbit immunoglobulin G.     

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.     

Trapping of reactive intermediates in enzymology. Exogenous flavin reduction during catalytic turnover of substrate by glyoxalase I

Although but weak inhibitors of glyoxalase I under steady-state conditions, flavins are reduced by yeast glyoxalase I (lactoyl-glutathione lyase, EC 4.4.1.5) plus its substrate (the hemithiolacetal from glutathione and phenylglyoxal) during catalytic turnover. Studies with 10-ethylisoalloxazine showed that this flavin reduction was peculiar not merely to glyoxalase I's substrate, but was characteristic of the complete system, enzyme plus substrate undergoing catalytic turnover. Flavins are poor hydride-ion acceptors and the reduction observed most likely represents an oxidative trap of a transient carbanion formed in the glyoxalase I mechanism of action. Hydrophobic flavins were more efficient traps than the hydrophilic ones, and values of the Km for the phenylglyoxal: glutathione hemithiolacetal adduct measured by the flavin-reaction and by normal steady-state kinetics were closely similar. This argues that trapping has occurred of an enediolate ion (an enzyme-generated carbanion) still bound to glyoxalase I.     

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.     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.     

Unique regulation of glyoxalase I activity during osmotic stress response in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>: neither the mRNA nor the protein level of glyoxalase I increase under conditions that enhance its activity

Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, to S-D-lactoylglutathione. The activity of glyoxalase I in the fission yeast Schizosaccharomyces pombe was increased by osmotic stress induced by sorbitol. However, neither the mRNA levels of its structural gene nor its protein levels increased under the same conditions. Cycloheximide blocked the induction of glyoxalase I activity in cells exposed to osmotic stress. In addition, glyoxalase I activity was increased in stress-activated protein kinase-deficient mutants (wis1 and spc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast.     

Characterization of glyoxalase I (E. coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Purification and partial characterization of glyoxalase I from bovine brain

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.     

Characterization of Glyoxalase I (E. coli)—Inhibitor Interactions by Electrospray Time-of-Flight Mass Spectrometry and Enzyme Kinetic Analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Sexual response in Saccharomyces cerevisiae: alteration of enzyme activity in the glyoxalase system by mating factor

Glyoxalase I activity in alpha-type budding yeast of the Saccharomyces cerevisiae strain was increased by exposure of alpha-type cells to supernatant of a culture of a-type yeast cells, although glyoxalase II activity was decreased by the same treatment. The alteration of enzyme activity in the glyoxalase system occurred during the 30-60 min period after exposure of alpha-type cells to a-type culture supernatant. No change of glyoxalase I and II activities was found in the case of the alpha-type strain, S. cerevisiae VQ3 (alpha ste3-1), which is deficient in a-factor receptors.