植物多酚氧化酶的研究进展

多酚氧化酶(polyphenol oxidase,PPO)是一类普遍存在于植物、真菌和昆虫质体中,由核基因编码,能与铜相结合的金属蛋白酶.它能分别催化单酚羟基和二羟基酚氧化为O-二酚和O-醌.植物多酚氧化酶是许多果蔬等农产品酶促褐变的主要原因,同时它在植物的光合作用、抗病虫害、生长发育以及花色的形成中起一定作用.本文综述了植物多酚氧化酶在细胞学、分子遗传学及其生产应用等方面的研究进展.     

植物多酚氧化酶的研究进展

多酚氧化酶(polyphenol oxidase, PPO)是一类普遍存在于植物、真菌和昆虫质体中,由核基因编码, 能与铜相结合的金属蛋白酶。它能分别催化单酚羟基和二羟基酚氧化为O-二酚和O-醌。植物多酚氧化酶是许多果蔬等农产品酶促褐变的主要原因, 同时它在植物的光合作用、抗病虫害、生长发育以及花色的形成中起一定作用。本文综述了植物多酚氧化酶在细胞学、分子遗传学及其生产应用等方面的研究进展。     

Characterization of mercapturic acid and glutathionyl conjugates of benzo[a]pyrene-7,8-dione by two-dimensional NMR.

Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine, and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dione and either N-acetyl-L-cysteine or glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[a]pyrene (a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[a]pyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[a]pyrenyl-peptide conjugate.     

A (1)H NMR study of the interaction of antitumor metallocenes with glutathione

The interaction of the antitumor active metallocene dihalides Cp(2)MCl(2) (M=Ti, Nb, Mo) and 1 equiv. of glutathione was studied by (1)H NMR spectroscopy at pD 2-7 in 4 mM NaCl solutions. No interaction between glutathione and titanocene dichloride was detected at pD 2, while at pD 5-7 competitive hydrolysis of the cyclopentadienyl ligands occurred. With niobocene dichloride formation of approximately 20% of an adduct was observed at pD 2 and 5, but hydrolysis of the Cp ligands in the adduct occurred over 24 h. Molybdocene dichloride formed two stable adducts at pD 6 which were tentatively assigned as a Cp(2)Mo-glutathione chelate involving coordination of the cysteine thiol and glycine carboxylate to the metal centre, and a thiol centred 1:2 Cp(2)Mo-glutathione complex. The implications for the mechanism of antitumor action of the metallocene dihalides is discussed.     

Polycyclic aromatic hydrocarbon (PAH) ortho-quinone conjugate chemistry: kinetics of thiol addition to PAH ortho-quinones and structures of thioether adducts of naphthalene-1,2-dione.

Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of an NADP+ dependent oxidation of non-K-region trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase (EC 1.3.1.20). Since these PAH o-quinones could be detoxified by non-enzymatic or enzymatic conjugation with cellular thiols, their reactivity with 2-mercaptoethanol, cysteine and glutathione (GSH) was examined by ion-pair reverse phase high pressure liquid chromatography (RP-HPLC). Second-order rate constants for the addition of these thiols to naphthalene-1,2-dione (NPQ) in water ranging from 4.9 x 10(3) - 1.1 x 10(4) min-1 M-1 and the reactions were complete within 10 min. When these reactions were conducted at near physiological pH (50 mM potassium phosphate buffer pH 7.0), the rate constants increased by 2-orders of magnitude. When benzo[a]pyrene-7,8-dione (BPQ) was substituted in these reactions the second-order rate constants decreased by 2-3 orders of magnitude and the reactions took several hours to reach completion. The decrease in reactivity can be explained by the presence of the bay region in BPQ. Methylation influenced the reactivity of PAH o-quinones with GSH and the following order of reactivity was observed: 7,12-dimethyl-benz[a]anthracene-3,4-dione (7,12-DMBAQ) > 12-methyl-BAQ, 7-methyl-BAQ and BAQ > BPQ. Of these quinones 7,12-dimethyl-BAQ was almost equi-reactive with NPQ. This suggests that methyl substitution in the bay and peri regions enhances reactivity with GSH. Using NPQ as a model for other PAH o-quinones, N-acetyl-L-cysteine, L-cysteine and GSH conjugates of NPQ were synthesized and characterized by [1H]- and [13C]NMR. Evidence for Michael type 1,4-addition products was obtained in which the resultant adduct could exist as either a catechol or o-quinone. By contrast, L-cysteine was able to form adducts via S- or N-attack and N-attack gave a purple p-iminoquinone. There was no evidence for the formation of bis-N-acetyl-L-cysteinyl-, bis-glutathionyl adducts or phenolic coupled products. The toxicity of thiol conjugates of NPQ remains to be explored.     

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein.     

Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, γ -glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine–synthase complex.     

Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine

19F NMR spectroscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N alpha-acetyl-N epsilon-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by 19F and 13C NMR spectroscopy and mass spectrometry. N alpha-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation was greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However N alpha-acetyllysine, at concentrations of greater than 100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Both stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated nephrotoxicity.     

Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase. A potential route of proximate carcinogen metabolism

The homogeneous dihydrodiol dehydrogenase of rat liver cytosol catalyzes the NADP-dependent oxidation of polycyclic aromatic trans-dihydrodiols, a reaction that may suppress their carcinogenicity provided the products of the reaction are noncarcinogenic. This report demonstrates that the products of naphthalene and benzo[a]pyrene trans-dihydrodiol oxidation are electrophilic o-quinones, which arise via autoxidation of catechols produced from the dihydrodiols by the action of dihydrodiol dehydrogenase. Oxidation of the trans-1,2-dihydrodiol of naphthalene or the 7,8-dihydrodiol of benzo[a]pyrene by the homogeneous rat liver dehydrogenase in 50 mM glycine at pH 9.0 led to the formation of multiple products by TLC, none of which co-migrated with the corresponding o-quinone standards. An identical result was obtained when these standards were incubated with buffer alone, suggesting that o-quinones were formed enzymatically from the dihydrodiols, and then underwent addition reactions with the glycine buffer. In subsequent reactions, the o-quinones formed from the enzymatic oxidation of the trans-dihydrodiols of naphthalene and benzo[a]pyrene were trapped by conducting the reactions in phosphate buffer containing 2-mercaptoethanol. The products of these reactions were identified by 500 MHz nmr and electron impact mass spectrometry as adducts of the 1,2-quinone of naphthalene (m/e M+ = 234) and the 7,8-quinone of benzo[a]pyrene (m/e M+ = 358), which contained mercaptoethanol as a thioether at C-4 and C-10, respectively. Kinetic analysis of the reactivity of the 1,2-quinone of naphthalene showed that the cellular nucleophiles, cysteine and glutathione, react very rapidly with the quinone. The 7,8-quinone of benzo[a]pyrene also reacted with glutathione and cysteine to form water-soluble metabolites, but did not react with adenosine or guanosine. These results suggest that o-quinones formed by enzymatic dihydrodiol oxidation may be effectively scavenged by cellular nucleophiles, resulting in their detoxification.     

Protein and nonprotein cysteinyl thiol modification by N-acetyl-p-benzoquinone imine via a novel ipso adduct.

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP), can arylate and oxidize protein and nonprotein thiols in the pathogenesis of APAP-induced hepatotoxicity. We report the first direct evidence for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tandem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposition kinetics of the GSH-NAPQI ipso adduct and product ratios suggested that the ipso adduct was readily reversible back to NAPQI under neutral and basic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either oxidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose catalytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provides significant new insights into possible reactions of quinone imines with cellular peptides and proteins.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Michael addition of thiols with 4-methyleneglutamic acid: Preparation of adducts,their properties and presence in peanuts

The thioethers, S-(4-amino-2,4-dicarboxybutyl)cysteamine, S-(4-amino-2,4-dicarboxybutyl)cysteine and S-(4-amino-2,4-dicarboxybutyl)glutathione, were synthesized by a Michael addition between 4-methyleneglutamic acid and the respective thiol. In dilute aqueous solution, the reactions exhibit second order kinetics; glutathione reacts much slower than cysteine or cysteamine. The adducts were characterized chromatographically, electrophoretically, and by their infra-red and nuclear magnetic resonance spectra. None of these thioethers was detected in peanut plants (Arachis hypogaea L.), even though large amounts of 4-methyleneglutamic acid, its amide, and glutathione are synthesized during peanut germination.     

o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their alpha-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The in-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their alpha-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from beta-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.     

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

Summary. The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO₂, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.     

Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial waste-water samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.     

Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants

Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insect-derived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities.     

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.     

Subcellular distribution of glutathione and cysteine in cyanobacteria

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.     

Thiol-glyoxylate adducts as substrates for rat kidney L-α-hydroxy acid oxidase

Rat kidney L-α-hydroxy acid oxidase (EC 1.1.3.15) catalyzes a rapid O₂ uptake at pH 7.5 when both glyoxylate and one of a number of various thiols are present. Thiols which are reactive in this system include: ethanethiol, 1-propanethiol, 2-mercaptoethanol, N-acetylcysteamine, propane-1,3-dithiol, dihydrolipoic acid, Coenzyme A, dephospho Coenzyme A, phosphopantetheine, and pantetheine. Notable physiological thiols that are not very reactive include: glutathione, L-cysteine and cysteamine. Presumably the substrate is a thiol-glyoxylate adduct because both the thiol and glyoxylate must be present in order to obtain a rapid enzyme-catalyzed reaction and oxalyl thioesters are the products of the enzymic reactions. Kinetic studies indicate that some of these adducts are better substrates than any others presently known. These and other results imply that a thiol-glyoxylate adduct may be the physiological substrate for L-α-hydroxy acid oxidase. A possible function for this reaction in metabolic control, mediated either by the oxalyl thioester or by oxalate, is briefly considered.     

Chemical and enzymic oxidation by tyrosinase of 3,4-dihydroxymandelate.

Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.