Kinetic mechanism and specificity of bovine milk sulphydryl oxidase

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.     

Resolution of sulphydryl oxidase from gamma-glutamyltransferase in bovine milk by covalent chromatography on cysteinylsuccinamidopropyl-glass.

1. Sulphydryl oxidase from bovine milk was purified by covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. Selective immobilization of the oxidase occurs through formation of a mixed disulphide between the enzyme and the substrate cysteinyl-glass matrix. Reductive elution of the bound protein can be accomplished with small thiols such as reduced glutathione (GSH), dithiothreitol or cysteine. This method leads to approx. 4000-fold purification of the enzyme from whey. Furthermore, complete resolution of sulphydryl oxidase from gamma-glutamyltransferase was achieved with this procedure. 2. Antibodies prepared against this purified enzyme quantitatively precipitated 95% of the GSH-oxidative activity from detergent-solubilized skim-milk membranes, whereas 100% of the transferase activity remained in the supernatant fraction; these findings confirmed the distinction between these two enzymes. 3. Reverse-phase high-pressure-liquid-chromatographic analyses of assay mixtures containing both enzymes revealed an array of GSH derivatives generated by a combination of the oxidative and hydrolytic activities. However, purified sulphydryl oxidase yielded only GSSG with concomitant stoichiometric loss of GSH. 4. The chromatographic method described is simple and reproducible, and may be applicable to isolation of sulphydryl oxidase from other tissues.     

Substrate specificity of cysteine lyase]

Substrate specificity is studied of cysteine lyase, a phosphopyridoxal-dependent enzyme belonging to the subgroup of beta-replacing lyases. This enzyme has a narrow specificity for the amino substrate; its only primary substrate is L-cysteine. Cysteine lyase has a broad specificity for the cosubstrate (replacing agent), catalysing the synthesis of L-cysteic acid from L-cysteine and sulfite ion or cystein thioesters (in the presence of some thiols). Enzyme is incapable to use alpha-phenyl- and alpha-methylcysteine as substrates. It is found that enzyme catalyses the exchange of alpha-H atoms of the aminoacid substrate cysteine with 3H2O. It does not catalyse alpha-hydrogenexchange in close structural analogues of substrate: L-alanine, D-serine, treonine, allo-threonine and 3-phosphoserine. L-Serine inhibited the synthesis of S-hydroxyethylcystein from cysteine and beta-mercaptoethanol (Ki of L-serine is 0,8-10(-2) M), participating at the first stage of reaction: the formation of a pyridoxylidenic derivative, which does not undergo the further alpha,beta-elimination of beta-replacement reactions.     

Xanthine oxidase activity of arthrobacter x-4 cells immobilized in glutaraldehyde-crosslinked gelatin

Summary Cells of Arthrobacter X-4 were immobilized by entrapment in gelatin crosslinked with glutaraldehyde. The xanthine oxidase activity and stability were determined at various temperatures. In comparison with bovine milk xanthine oxidase the bacterial enzyme is more stable and has a different substrate specificity. 1-Methylxanthine was oxidized on a preparative scale.     

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of -glucose, and the product of -glucose oxidation is -gluconic acid.

The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k_cat is 20, k_ox 11 and k_red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used.

The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.     

Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.     

Oxidase of the white rot fungus Panus tigrinus 8/18

Extracellular oxidase of the white rot fungus Panus tigrinus earlier reported as laccase)contains copper but has no absorption spectrum typical of ‘blue’ oxidases. Thioglycolate and sodium azide inhibit the activity of this enzyme at concentrations 2.5–3 orders lower than those needed for fungal laccases. The oxidase of P. tigrinus oxidizes syringaldazine, coniferyl alcohol, ABTS, syringic acid, diaminobenzidine, guaiacol, catechol and vanillylacetone with different efficiencies. Oxygen consumption and no hydrogen peroxide formation were detected during substrate oxidation by P. tigrinus oxidase. It is proposed that P. tigrinus oxidase is a new ligninolytic enzyme.     

Skin sulfhydryl oxidase. Purification and some properties

A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol of by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, D-penicillamine, and L-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 +/- 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.     

Production of novel oligosaccharide oxidase by wheat bran solid-state fermentation

A search for oxidases that catalyze the oxidation of oligosaccharides has resulted in the isolation of several soil-derived fungus strains which produced novel oligosaccharide oxidases with different substrate specificity on wheat bran solid culture. One of these oxidases produced by Acremonium strictum T1 strain has been characterized. This enzyme showed high reactivity toward maltose, lactose, cellobiose and maltooligosaccharides composed of up to seven glucose units, and was named as glucooligosaccharide oxidase based on its substrate specificity. Strain T1 was subjected to a strain improvement program, and an enzyme hyper-producing mutant strain T1-38 was selected. This mutant strain produced glucooligosaccharide oxidase 75 times higher than the wild type strain T1. When cultivated in a solid medium comprised of 1 part of wheat bran and 1 part of water (w/w), enzyme activity reached a maximum level of 6 units per g of culture medium after 4 days cultivation. Characteristics of the enzyme including the substrate specificity were compared with two other novel oligosaccharide oxidases isolated in this laboratory. Batch type conversion of lactose to lactobionic acid using crude enzyme was also discussed.     

Isolation and characterization of catechol oxidase from Solanum melongena

Catechol oxidase was distributed in soluble and particulate fractions of Solanum melongena. The purified preparation appears to be homogeneous by polyacrylamide gel electrophoresis. The enzyme shows two pH maxima—with catechol, 6.5 and 7.5; and with dopa, 6.5 and 7.9. The latent form of the enzyme does not occur in S. melongena. The preparation resembles the enzyme from other sources in substrate specificity towards various mono- and diphenols, having a higher affinity for catechol than dopa; this tendency increases on purification. The cresolase activity decreases with purification and a lag period with p-cresol is observed. The oxidation of mono- and diphenols is inhibited by ascorbic acid, sulphydryl compounds and chelating agents.     

Defining substrate specificity and catalytic mechanism in ascorbate peroxidase

Haem peroxidases catalyse the H2O2-dependent oxidation of a variety of, usually organic, substrates. Mechanistically, these enzymes are very well characterized: they share a common catalytic cycle that involves formation of a two-electron oxidized intermediate (Compound I) followed by reduction of Compound I by substrate. The substrate specificity is more diverse, however. Most peroxidases oxidize small organic substrates, but there are prominent exceptions to this and the structural features that control substrate specificity remain poorly defined. APX (ascorbate peroxidase) catalyses the H2O2-dependent oxidation of L-ascorbate and has properties that place it at the interface between the class I (e.g. cytochrome c peroxidase) and classical class III (e.g. horseradish peroxidase) peroxidase enzymes. We present a unified analysis of the catalytic and substrate-binding properties of APX, including the crystal structure of the APX-ascorbate complex. Our results provide new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase-mediated catalysis for more than 20 years.     

Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

The histochemistry of thiols and disulphides. III. Staining patterns in rat tissues

Synopsis With the aid of new staining methods, thiol groups produced by the reduction of disulphide bonds were positively distinguished from pre-existing groups in paraffin sections of several organs of the rat. Good preservation of structures in which the natural thiol-disulphide balance had been maintained was sought by fixing the tissues in neutral formalin containing an organomercurial. After dissociation of the resulting mercaptide bonds that protected the native thiols, these were shown in one colour and then disulphide sites in another within the same sections. Intracellular granules and extracellular membranes rich in disulphides thereby stood out in red against the predominantly blue labelling of the cellular ground plasm. Intimate mixtures of the two forms in some places and the presumed transformation of thiols to disulphides in others, notably the keratinizing epithelium of the tongue, were readily seen. Supplemented by separate visualization of thiols and disulphides along with suitable controls for specificity of staining, the results obtained diverged in some major respects from those of previous investigations.     

Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water-in-oil microemulsion system

1. Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water-in-oil microemulsion, which consists of discrete nanometer-diameter water droplets dispersed in a continuous water-immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface. 2. Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule. 3. Studies are presented on the enzyme-catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations. 4. The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis-Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.     

Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.     

The formation of mixed disulphides in rat lung following paraquat administration Correlation with changes in intermediary metabolism

We have investigated the hypothesis that the formation of mixed disulphides between protein sulphydryl and glutathione may be responsible for controlling the activity of the pentose phosphate pathway and fatty acid synthesis in rat lung. Using lung slices, taken from rats 2 h after dosing with a range of concentrations (5–80 mg/kg) of the pulmonary toxin paraquat, the pentose phosphate pathway was found to be stimulated in direct proportion to a reduction in fatty acid synthesis. These effects were also linearly related to an increase in mixed (total) disulphide levels in the lung. This was quantitatively similar to an increase in mixed (glutathione) disulphides, although non-protein sulphydryl and oxidised levels remained normal. Thus, an early biochemical event in the mechanism of paraquat toxicity in the lung involves an increased formation of mixed (glutathione) disulphides and simulatneous regulation of pentose phosphate pathway activity and fatty acid synthesis. These data support the concept that the formation of mixed disulphides of protein and glutathione is a mechanism for maintaining NADPH levels despite the ‘redox’ stress caused by the cyclical and NADPH dependent reduction and reoxidation of paraquat.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase.

1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3) NH, (9) N or (3) N, (9) NH. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.     

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B

Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.