Showing papers on "Lactoylglutathione lyase published in 1975"

A glyoxalase I inhibitor of a new structural type produced by Streptomyces.

Abstract: Many streptomyces strains produced an inhibitor of crude glyoxalase prepared from rat liver which did not inhibit glyoxalase I prepared from yeast. Another inhibitor, C11H14O6, which inhibited glyoxalases prepared from both rat liver and yeast was obtained from a cultured broth of Streptomyces griseosproeus and crystallized. Preincubation of this inhibitor with reuduced glutathione increased its inhibitory activity, which suggested its reaction with reduced glutathione. It showed a strong inhibition of growth of HeLa cells and inhibition of Ehrlich ascites carcinoma by daily injection. It also showed weak inhibition of the solid type of Ehrlich carcinoma and prolonged the survival period of mice inoculated with L-1210 cells.

Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.

Abstract: The disproportionation of alpha-ketoaldehydes, catalyzed by yeast glyoxalase I, has been reported to involve a random pathway mechanism where one branch utilizes the hemimercaptal of glutathione and the alpha-ketoaldehyde in a one-substrate pathway, and the other branch utilizes first glutathione and then the alpha-ketoaldehyde in an ordered two-substrate pathway. The relative importance of the two pathways has been evaluated at 5 degrees in the pH range 3-7, using methylglyoxal and phenylglyoxal as representative aliphatic and aromatic alpha-ketoaldehydes, by comparing initial rates of hemimercaptal formation in the absence of enzyme with initial rates of product formation in the presence of high enzyme concentrations. If the enzyme is not added last, the initial rates of product formation are the same as the initial rates of adduct formation even under conditions where it could be shown that dehydration of the hydrated alpha-ketoaldehyde is not entirely rate determining. If the enzyme is added after hemimercaptal formation, there is a "burst" of product formation equivalent to the amount of hemimercaptal, followed by a slower reaction, consistent with the one-substrate pathway. Additional support for this pathway was obtained from a study of the effects of added thiol reagents on the "burst" kinetics. The broad specificity of yeast glyoxalase I for both aliphatic and aromatic alpha-ketoaldehydes, reflected in Vmax values which are insensitive to the nature of the alpha-ketoaldehyde drops abruptly if the side chain of the alpha-ketoaldehyde is sterically crowded. The hemimercaptal of tert-butylglyoxal has a Vmax 300-fold smaller than Vmax for methylglyoxal; 2,4,6-trimethylphenylglyoxal is essentially inactive as a substrate even though the closely related compound 2,4-dimethylphenylglyoxal is a normal substrate. Analysis of the Vmax and Km (or Ki) values of these alpha-ketoaldehydes suggests that sterically crowded side chains affect both enzyme-substrate formation and the catalytic reaction.

The structure of a glyoxalase I inhibitor and its chemical reactivity with SH-compounds.

Abstract: The structure of a glyoxalase I inhibitor (I), isolated from a cultured broth of Streptomyces griseosporeus, was found to be 2-crotonyloxymethyl-4,5,6-trihydroxy-cyclohex-2-enone by chemical studies. Stereochemistry and absolute configuration were determined to be 4R, 5R and 6R by X-ray crystallographic analysis of a bromine-containing crystalline derivative. The crotonyloxy group of I shows a surprising proclivity to be displaced by SH-compounds. This property is shown to be the basis for its biological activity.

Purification and Properties of Glyoxalase I from Sheep Liver

Abstract: 1 Glyoxalase I has been obtained in electrophoretically pure form from sheep liver by a procedure which includes ammonium sulfate and poly(ethylene glycol) fractionations and column chromatographies on hydroxyapatite, Cibacron Blue–Sephadex G-100 and DEAE-cellulose. The specific activity of the homogeneous preparations is about 4000 units/mg of protein (25° C). Three separate peaks of activity were obtained in the last column on DEAE-cellulose (DE-32). No signs of heterogeneity were seen in the previous steps. Purified but not crude preparations gave two activity peaks on disc gel electrophoresis. 2 The isoelectric point of metal-free apoglyoxalase I is 5.0 by isoelectric focusing. The apparent molecular weight of glyoxalase I is 45 900 from gel chromatography. From dodecylsulfate gel electrophoresis the enzyme has a subunit molecular weight of 21000. 3 In addition to methylglyoxal, phenylglyoxal and kethoxal are good substrates of sheep liver glyoxalase I. Hydroxypyruvaldehyde and glyoxal react more slowly. 4 Catalytically inactive apoenzyme of sheep liver glyoxalase I has been prepared by dialysis against EDTA and Chelex-100. All of the activity is restored by Mg2+ ions; Zn2+, Mn2+, Co2+, Ni2+ and Ca2+ ions, in decreasing order of maximum velocity, give partial reactivation. Of these metals, Mg2+ has the highest (2.8 mM) and Zn2+ the lowest (0.007 mM) apparent half-saturation concentration when assayed in 80 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (Hepes) buffer pH 6.8. 5 Several chelating agents are inhibitors of sheep liver glyoxalase I. In all cases Mg2+ reverses the inhibition after a short incubation time.

[104] Methylglyoxal synthase

Abstract: Publisher Summary This chapter discusses the assay method, purification procedure, and properties of methylglyoxal synthase Methylglyoxal production can be measured continuously by coupling it to the lactoylglutathione lyase reaction and monitoring the formation of S -lactoylglutathione at 240 nm (Method 1); or discontinuously, at the end of the reaction period, by measurement of the bis-derivative formed on reaction with the 2,4-dinitrophenylhydrazine reagent (Method 2) The purification procedure of E coli and P saccharophila enzymes are also tabulated The enzyme in crude extracts loses little activity when stored at 0–4° over several days The most pure enzyme preparation (specific activity 530) shows one major and two minor protein bands 1° on acrylamide gel electrophoresis at pH 89 Neither dihydroxyacetone nor Da-glyceraldehyde 3-phosphate could substitute for dihydroxyacetone phosphate in the reaction The E coli and P saccharophila enzymes have molecular weights of approximately 67,000 as estimated by gel filtration Methylglyoxal synthase has been found in various Enterobacteriaceae and in certain Enterobacteriaceae-like organisms, such as Aeromonas formicans and Obesumbacterium proteus It has also been found in the strict anaerobes Clostridium pasteurianum and Clostridium tetanomorphum

The Structure of MS-3: A Glyoxalase I Inhibitor Produced by a Mushroom

Abstract: The structure of MS–3, a glyoxalase I inhibitor produced by a mushroom, was established to be 3′,4′-dihydroxymethyl-5′-hydroxy-6′-(3-methyl-2-butenyl)-phenyl-2,4-dihydroxy-6-methyl-benzoate.

Growth inhibitory properties of aromatic alpha-ketoaldehydes toward bacteria and yeast. Comparison of inhibition and glyoxalase I activity.

Abstract: The alpha-ketoaldehydes methylglyoxal and substituted phenylglyoxals are similar in their abilities to inhibit the growth of Escherichia coli and yeast. When logarithmically growing cells are added to media containing 0.3-1 mM alpha-ketoaldehyde, growth stops for several hours, after which normal growth resumes. The period of growth inhibition does not appear to show any correlation with the ability of glyoxalase I to detoxify these alpha-ketoaldehydes. E. coli and yeast glyoxalase I show markedly different substrate specificities. For example, although both enzymes show broad specificity for both aliphatic and aromatic alpha-ketoaldehydes, 2,4,6-trimethylphenylglyoxal is a substrate for the E. coli enzyme but not for the yeast enzyme. Nevertheless, this alpha-ketoaldehyde inhibits the growth of both E. coli and yeast, similar to the other alpha-ketoaldehydes. Enzymes other than glyoxalase I must play a major role in the metabolism of these alpha-ketoaldehydes during the period of growth inhibition.

Inactivation of Glyoxalase I from Porcine Erythrocytes and Yeast by Amino‐Group Reagents

Abstract: Glyoxalase I from porcine erythrocytes and from yeast is inactivated by the amino-group reagents 1-fluoro-2,4-dinitrobenzene, 5-dimethylaminonaphthalene-1-sulfunyl chloride, and 2,4,6-trinitrobenzenesulfonate (N3ph-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 N3ph-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 · 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 dervatives, 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.

Synthesis and study of glutaryl-S-(omega-aminoalkyl)-L-cysteinylglycines as inhibitors of glyoxalase I.

Abstract: This thesis describes the synthesis and preliminary enzymatic study of glutaryl-S-(8-aminooctyl)-L-cysteinylglycine and glutaryl-S-(10-aminodecyl)-L-cysteinylglycine as inhibitors of glyoxalase I. These analogs of glutathione were prepared as potential ligands for affinity chromatography purification of glyoxalase I. The compounds were synthesized by a seven-step procedure in overall yields of 24% for the octyl analog and 33% for the decyl analog. Both compounds exhibited mixed type inhibition of the enzyme, with the decyl derivative being more inhibitory than the octyl derivative. The inhibition was nonlinear (parabolic) for both compounds. Although less inhibitory than the corresponding S-substituted glutathione derivatives, these analogs are promising candidates for affinity chromatography ligands. Such compounds may also be useful in studying the mechanism of glyoxalase I.

Synthesis and study of glutaryl-s-(omega-aminoalkyl)-l-cysteinylglycines as inhibitors of glyoxalase i

Abstract: This thesis describes the synthesis and preliminary enzymatic study of glutaryl-S-(8-aminooctyl)-L-cysteinylglycine and glutaryl-S-(10-aminodecyl)-L-cysteinylglycine as inhibitors of glyoxalase I. These analogs of glutathione were prepared as potential ligands for affinity chromatography purification of glyoxalase I. The compounds were synthesized by a seven-step procedure in overall yields of 24% for the octyl analog and 33% for the decyl analog. Both compounds exhibited mixed type inhibition of the enzyme, with the decyl derivative being more inhibitory than the octyl derivative. The inhibition was nonlinear (parabolic) for both compounds. Although less inhibitory than the corresponding S-substituted glutathione derivatives, these analogs are promising candidates for affinity chromatography ligands. Such compounds may also be useful in studying the mechanism of glyoxalase I.