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Glyoxalase I (EC 4.4.1.5) was purified about 10000-fold from pig erythrocytes in a yield of approx. 20%. The purification included affinity chromatography on S-hexylglutathione coupled to Sepharose 4B. The purified enzyme normally contained two catalytically active components which were resolved by polyacrylamide-gel electrophoresis. After treatment with reduced glutathione only one component was found. The two components were also demonstrable after isoelectric focusing or DEAE-cellulose chromatography and could also in these cases be fused into one species by preincubation with reduced glutathione. It is proposed that the most acidic form of glyoxalase I is a mixed disulphide with glutathione. Except for these interconvertible forms, the purified enzyme was homogeneous, as judged by disc electrophoresis and sodium dodecyl sulphate/polyacrylamidegel electrophoresis. The molecule is a dimer (48000 daltons), composed of apparently identical subunits (24000 daltons). The isoelectric point was 4.8 at 4 degrees C. The amino acid composition was consistent with the low isoelectric point. The enzyme contained about two thiol groups per enzyme molecule. EDTA inactivated the enzyme and bivalent metal ions could restore fully or partially the catalytic activity; Mg(2+) and Mn(2+) gave highest activity. It is proposed that a major biological function of glyoxalase I is the detoxification of methylglyoxal formed by enterobacteria in the alimentary canal.

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 K(m) 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 0.08mum for the Zn-containing enzyme to 1.3, 1.7 and 2.4mum 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 (6.9mum). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn(2+)-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.

Glyoxalase I from porcine erythrocytes and from yeast is inactivated by the amino-group reagents 1-fluoro-2,4-dinitrobenzene, 5-dimethylaminonaphthalene-1-sulfonyl chloride, and 2,4,6-trinitrobenzenesulfonate (N-3ph-S). The inactivation follows pseudo-first-order kinetics, and the apparent first-order rate constant increases with pH, indicating that the basic form of a nucleophilic group is modified. The effect of increasing the inactivator concentration was tested with N-3PH-S, and it was found that the apparent rate constant increased to a limiting value. Such a result is consistent with a mechanism involving formation of a reversible inactivator x enzyme complex prior to the actual inactivation. Experiments with erythrocyte glyoxalase I and a variety of sulfhydryl-group reagents failed to show a dependence on sulfhydryl groups for catalytic activity, in contrast to previous results with the yeast enzyme. These experiments seem to exclude the possibility that essential sulfhydryl groups of the erythrocyte enzyme are modified by the amino-group reagents. Failure of reactivation of yeast glyoxalase I, and the similarities with the erythrocyte enzyme suggest that yeast glyoxalase I is not modified at essential sulfhydryl groups either by the latter reagents. This assumption has further support from experiments involving simultaneous inactivation with amino and sulfhydryl-group reagents. The results are consistent with the interpretation that amino groups of glyoxalase I are essential for catalytic activity. Glutathione derivatives, which are reversible competitive inhibitors of glyoxalase I, were found to protect the enzyme against inactivation by amino-group reagents. However, the concentration required for half-maximal protection was considerably higher than the inhibition constant of the reversible inhibition, which indicates that at least two molecules of the protector must be bound to the enzyme before full protection is obtained.

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)

X-ray edge and extended absorption fine structure spectra of Zn2+ at the active site of glyoxalase I have been measured. The edge spectrum reveals a simple set of transitions consistent with a 7-coordinate or distorted octahedral Zn2+ model complex. Analysis of the fine structure rules out sulfur ligands to Zn2+ and yields a best fit complex with Zn2+-N (or Zn2+-O) distances of 2.04 and 2.10 A, which are too great for tetrahedral Zn2+ coordination but are appropriate for an octahedral or more highly coordinated complex. Peaks of electron density in the Fourier-transformed region of the higher order shells at distances of 3-4 A from the Zn2+-imidazole model similar to those found with known Zn2+-imidazole model complexes, including carbonic anhydrase [Yachandra, V., Powers, L., & Spiro, T.G. (1983) J. Am. Chem. Soc. 105, 6596-6604], indicating at least two imidazole ligands to Zn2+ on glyoxalase I. Binding of the heavy atom substrate analogue S-(p-bromobenzyl)glutathione did not significantly alter the number of atoms directly bonded to Zn2+ or their distances. No evidence for coordination of the cysteine sulfur of glutathione by the Zn2+ was obtained, and no heavy atom signal from bromine was detected, indicating this atom to be greater than or equal to 4 A from the Zn2+. However, conformational changes of the imidazole ligands of Zn2+ upon binding of the substrate analogue were suggested by changes in the relative intensity of the doublet peaks at 3-4 A from the Zn2+ and assignable to imidazole.(ABSTRACT TRUNCATED AT 250 WORDS)

The apoenzyme of glyoxalase I (EC 4.4.1.5) from human erythrocytes was prepared by removal of Zn2+ with ethylenediaminetetraacetic acid (EDTA). Methanol was used as a stabilizing agent. Extended dialysis was required to remove EDTA from the resulting solution of apoenzyme. Reconstitution with Mn2+ was followed by measuring enzyme activity, electron paramagnetic resonance of free Mn2+ ions, and nuclear magnetic resonance of water protons. The holoenzyme contained two Mn2+ per protein dimer and had approximately 50% of the catalytic activity of the native enzyme. The binding of the cosubstrate glutathione (gamma-L-glutamyl-L-cysteinylglycine), the product S-D-lactoyl-glutathione, and the competitive inhibitor S-(p-bromo-benzyl)glutathione was monitored by the quenching of the intrinsic tryptophan fluorescence and by the proton relaxation enhancement of water bound to Mn2+ in the active site of the enzyme. The dissociation constants were 1.1 mM, 0.42 mM, and 0.54 microM for glutathione, S-D-lactoylglutathione, and S-(p-bromobenzyl)glutathione, respectively. The temperature and frequency dependences of the longitudinal and transverse paramagnetic relaxation rates, 1/T1p and 1/T2p, were studied for water. The results were analyzed in terms of correlation and exchange times. In addition proton and deuteron relaxation rates were measured in parallel at two different magnetic fields. Good agreement between the two approaches of analysis was noticed. The data show that two water molecules are bound in the first coordination sphere of Mn2+ in the active site of glyoxalase I. When S-(p-bromobenzyl)glutathione or S-D-lactoylglutathione is bound to the enzyme, only one exchangeable water molecule could be detected, indicating occlusion of the second water molecule. An enediol mechanism involving the metal-bound water is proposed for the catalysis effected by glyoxalase I.

Metal dissociation constants for glyoxalase I from human erythrocytes were determined by use of nitrilotriacetic acid as a metal buffer. The constants for Zn2+, Co2+, Mn2+, and Mg2+ were (2.7 +/- 0.3) X 10(-11) M, (3.0 +/- 0.8) X 10(-10) M, (4.9 +/- 0.5) X 10(-9) M, and (1.0 +/- 0.2) X 10(-6) M, respectively, demonstrating that the natural cofactor, Zn2+, has the highest affinity for the apoprotein. The results are consistent with the proposal of nitrogen and oxygen atoms as ligands to the metal in the active site of glyoxalase I. In the application of the metal buffer technique, it was found that both 1:1 and 1:2 complexes of the metal ions and nitrilotriacetic acid have to be considered.

Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.

Glyoxalase I catalyzes the formation of S-D-lactoyl-glutathione via the hemimercaptal adduct of methylglyoxal and glutathione. This enzymatic reaction, which has been considered virtually irreversible, was found to be reversible under such conditions that glutathione liberated from the thiolester was trapped. The reverse reaction could be monitored spectrophotometrically by use of 5,5'-dithiobis-(2-nitrobenzoate). In addition to 5,5'-dithiobis-(2-nitrobenzoate), 2,2'-dithiobispyridine and cystamine were used to promote the reverse reaction. S-D-Lactoylglutathione did not hydrolyze in the presence of glyoxalase I under the conditions investigated, as shown by its stability in the absence of thioltrapping agents. Proof of the reversal of the reaction was obtained by demonstrating the formation of stoichiometric amounts of methylglyoxal and glutathione from S-D-lactoylglutathione. Catalysis of the reverse reaction was dependent upon the presence of a bivalent metal ion in the active site of the enzyme. Apoenzyme, obtained by removal of the essential Zn2+ from the active site, did not catalyze the reverse reaction, but catalytic activity was restored by addition of Zn2+, Mg2+, Mn2+, or Co2+. The reverse reaction was also catalyzed by glyoxalase I from yeast. Linear competitive inhibition (Ki = 0.64 mM) was obtained with 5,5'-dithiobis-(2-nitrobenzoate), which necessitated correction of the apparent kinetic parameters of the reverse reaction. The corrected values for the reverse reaction catalyzed by glyoxalase I from human erythrocytes with S-D-lactoylglutathione as substrate were kcat = 3.6 s-1 and Km = 1.9 mM. Combination of these values with the corresponding parameters for the forward reaction allowed calculation, through the Haldane relation, of the equilibrium constant, Keq = 1.1 X 10(4), for the isomerization between the hemimercaptal of methylglyoxal and glutathione and S-D-lactoylglutathione. The strong reversible competitive inhibitor of the forward reaction, S-p-bromobenzylglutathione, also inhibited the reverse reaction competitively (Ki = 0.38 microM).

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.

Co(II)-glyoxalase I has been prepared by reactivation of apoenzyme from human erythrocytes with Co2+. The visible absorption spectrum showed maxima at 493 and 515 nm and shoulders at 465 and 615 nm. The absorption coefficients at 493 and 515 nm were 35 and 33 M-1 cm-1/cobalt ion, respectively; i.e. 70 and 66 M-1 cm-1 for the dimeric metalloprotein. The product of the enzymatic reaction, S-D-lactoylglutathione, although binding to Co(II)-glyoxalase I, had no demonstrable effect on the visible absorption spectrum, indicating binding outside the first coordination sphere of the metal. The EPR spectrum at 3.9 K was characterized by g1 approximately 6.6, g2 approximately 3.0, and g3 approximately 2.5, and eight hyperfine lines with A1 = 0.025 cm-1. Binding of the strong competitive inhibitor S-p-bromobenzylglutathione to Co(II)-glyoxalase I gave three g values: 6.3, 3.4, and 2.5, indicating a conformational change affecting the environment of the metal ion. Both optical and EPR spectra strongly suggest a high spin Co2+ with octahedral coordination in the active site of the enzyme. The similarities in kinetic properties between native Zn(II)-glyoxalase I and enzyme substituted with Mg2+, Mn2+, or Co2+ is consistent with the view that these enzyme forms have the same metal coordination in the protein.

The paramagnetic effects of Mn2+ . glyoxalase I on the 13C relaxation rates of the reaction product, S-(D-lactoyl)glutathione, separately enriched in the lactoyl carbonyl (C-1) and hydroxymethylene (C-2) carbons, have been measured at 62.8 MHz. The 1/fT1p values of C-1 (1100 +/- 120 s-1) and C-2 (712 +/- 290 s-1) and the previously determined tau c (0.74 ns) yield Mn2+ to carbon distances of 5.7 +/- 0.3 and 6.1 +/- 0.5 A, respectively. These distances, together with previously determined Mn2+-proton distances (Sellin, S., Rosevear, P.R., Mannervik, B., and Mildvan, A.S. (1982) J. Biol. Chem. 257, 10023-10029) constrain the thioester carbonyl group of the product to point toward the metal, with the oxygen positioned to accept a hydrogen bond from a water ligand, in a kinetically competent, second sphere complex. Model-building studies indicate that any averaging of multiple second sphere complexes would require as a major contributor at least one conformation with the lactoyl carbonyl oxygen within hydrogen-bonding distance of an intervening water ligand. Such a structure would facilitate polarization of the carbonyl group in the reverse glyoxalase reaction.

The conformations of four derivatives of glutathione bound at the active site of the metalloenzyme glyoxalase I have been determined by NMR measurements and by computer model building using a distance geometry approach. Paramagnetic effects of Mn2+-glyoxalase I on the longitudinal relaxation rates of the carbon-bound protons of the substrate analog S-(acetonyl)-glutathione at three frequencies, the hydrophobic competitive inhibitor S-(propyl)glutathione at four frequencies, and the charged competitive inhibitor S-(carboxymethyl)glutathione at a single frequency were used to calculate Mn2+ to proton distances in each complex. These and previously determined distances from Mn2+ to the protons and 13C-enriched carbon atoms of the product S-(D-lactoyl)glutathione were used in a distance geometry program to compute the conformations of each enzyme-bound derivative which best fit the measured distances and other known constraints such as bond lengths, van der Waals radii, planar and trans-peptide bonds, and thioester linkages. The distance geometry program also provided a measure of the uniqueness of the conformations consistent with the experimental data. Extended Y-shaped conformations were detected for each of the bound glutathione derivatives, similar to the x-ray structure and the theoretically calculated conformation of glutathione itself, suggesting this to be a low energy form. Acceptable conformations of each enzyme-bound derivative fell into two classes with the metal either above or below the mean plane through the glutathione compound. The conformational uncertainty within each class was relatively small, ranging from deviations of 0.9-1.9 A in the average positions of each of the atoms. A small but significant difference in the conformation of the substrate analog as compared to the product was detected in the position of the reaction center carbon directly bonded to the glutathione sulfur atom. Unlike the second-sphere metal complexes formed by the bound substrate analog, the product, or the hydrophobic competitive inhibitor, the charged competitive inhibitor S-(carboxymethyl)glutathione binds farther from the metal, in the third coordination sphere.