Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii.

The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-alcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km = 0.84 mM, Vmax = 52 U/mg) to 4-methoxybenzyl alcohol (Km = 0.04 mM, Vmax = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.     

Metabolism of m-tert.-butylphenyl N-methylcarbamate in insects and mice

The metabolism of m-tert.-butylphenyl N-methylcarbamate was studied in mice and five species of insects. Both the tert.-butyl group and the N-methyl group were hydroxylated. The major phenolic metabolite was m-(beta-hydroxy-tert.-butyl)phenol, which was identified by mass spectroscopy. Significant amounts of dihydroxy compounds were formed at a constant rate from the start of the enzymic oxidation process. The considerable species variation in the yields of the different types of oxidation products suggests that N-demethylation and oxidation of the tert.-butyl groups were catalysed by different enzymes. A microsomal NADPH-dependent enzyme also catalysed the splitting of the ester link in the insecticide.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

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.     

Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane

1. Hypoxanthines, bearing at position 8 aryl or pyridyl substituents, are converted by bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) into the corresponding xanthines at low rates. Oxidation is accelerated considerably when the 8-pyridyl substituents are quaternised. 2. In the enzymic oxidation of quaternary 8-pyridylhypoxanthines a lag phase precedes the attainment of a constant, maximal reaction rate. It is assumed that the delay is due to a relatively slow conformational change in the active enzymic center. 3. In 8-(3'-N-methylpyridinio)xanthine betaine, also the pyridinium moiety is attacked at high pH (9-11) to yield an N-methyl-2-pyridone. The analogous pyridone is the only oxidation product of 1-methyl-8-(3'-N-methylpyridinio)-hypoxanthine betaine, which is not attacked in the pyrimidine ring. 4. The cationic substrates are attracted to the enzyme by an anionic group, which probably forms an ion pair with a protonated amino group in or near the active center.     

An essential carboxylic acid group in human prostate acid phosphatase.

Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and L-(+)-tartrate, but not in the presence of non-inhibitory D-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of D- and of L-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.     

Biodegradation of phenol by enzymes from Pseudomonas sp. immobilized onto ultrafiltration membranes

A method of immobilizing enzymes from Pseudomonas sp. that decompose phenol on polymeric ultrafiltration membranes is described. Transport-separation properties of neutral and enzymic membranes have been compared and the optimal ultrafiltration process parameters of a model phenol solution have been determined. The immobilized enzyme system was applied to the biodegradation of phenol in coke wastewaters.     

Oxidation of methyl derivatives of pteridin-4-one, lumazine and related pteridines by bovine milk xanthine oxidase.

1. Pteridin-4-ones, methylated at nitrogen or carbon, N-methylated lumazines and related oxopteridines were studied as substrates of a highly purified bovine milk xanthine oxidase (xanthine : oxygen oxidoreductase, EC 1.2.3.2). 2. The enzyme can oxidise at high rates both uncharged and anionic substrates. Variation of enzymic activity with pH is mainly due to pH-dependent changes in the active enzymic center. 3. Milk xanthine oxidases at different stages of purification convert pteridin-4-one into the 4,7-dione (compound 13 in this article). 4. Methylation at C-6 in the pyrazine moiety enhances enzymic attack at C-2 in the pyrimidine ring. N-Methylation may increase or reduce rates of oxidation. 5. For oxidation at C-2, the most favorable form of the substrate bears a double bond at C(2) = N(3). Attack at C-7 is enhanced strongly in structures bearing a double bond at C(6) = C(7). 6. In general, pteridines react with xanthine oxidase as non-hydrated molecules. However, oxidation of 8-methyllumazine at C-7 may take place by dehydrogenation of the 7-CHOH group of the covalently hydrated molecule.     

Effect of testosterone-estradiol-binding globulin in the enzymic oxidoreduction of 17-oxygenated c19 steroids.

The effect of both testosterone-estradiol-binding globulin (TeBG) and albumin on enzymic oxidoreduction of four 17-oxygenated C19 steroids by bacterial 17beta-hydroxysteroid:NAD oxidoreductase from Pseudomonas testosteroni was investigated. The decreased yields of products under presence of TeBG were found in both directions of reversible enzymic reaction. This finding was unexpected in the case of enzymic reduction in which the opposite effect could be assumed with respect of high affinity of the product but not the substrate to TeBG. Kinetically, the competition between enzyme and binding protein for the substrate occurs at enzymic oxidation, whereas the mechanism resembling non-competitive inhibition operates in the enzymic reduction.     

Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.     

Investigation of the arylnitroso reductase activity of pig liver aldehyde reductase.

The reduction of p-nitroso-N-dimethylaniline, p-nitroso-N-diethylaniline, p-nitrosophenol and p-nitroso-N-phenylaniline with NADPH in the presence of aldehyde reductases 1 and 2 is described. The reactivity of these nitroso substrates is increased by hydrophobic substituents and those promoting OH- elimination from the molecule of the reduced substrate. NN-Dimethylbenzoquinonedi-iminium cation was proved to be the reaction product formed from p-nitroso-N-dimethylaniline. The kinetics of the reduction of p-nitroso-N-dimethylaniline catalysed with aldehyde reductase 1 are rather complex at pH 7, and the preferred-pathway mechanism is probably involved. The reaction sequence approaches the ordered pattern at pH 8.5. It was shown that NADPH in equilibrium NADP+ recyclization proceeds in the presence of NADP+, p-nitroso-N-dimethylaniline, cyclohexanol and aldehyde reductase 1, the alcohol oxidation being the slowest step in this reaction. However, the rate of cyclohexanol oxidation surpasses that of the dissociation of NADPH from the enzyme.     

Mitochondrial malate dehydrogenase of bovine cerebrum. Characterization and mechanisms of inhibition by silver ions.

Attempts were made to characterize mitochondrial malate dehydrogenase [L-malate: NAD+ oxidoreductase, EC 1.1.1.37] (M-MDH) purified from bovine cerebrum and to elucidate the mechanisms responsible for inhibition of the enzymic activity by Ag+. The molecular weights of the native enzyme and its subunits were 54,000-55,000 and 30,000-32,000, respectively. In general, the physiochemical and catalytic properties of bovine cerebral M-MDH was not very different from those of other corresponding mammalian enzymes. Incubation of the enzyme with Ag+ caused the loss of equivalent amounts of sulfhydryls with a parallel decrease of the enzymic activity. When the enzyme was exposed to 2-, 3.5-, and 5-fold molar excesses of Ag+, the enzymic activity showed an initial rapid fall and a subsequent slow restoration to a partially inactivated level (60-70, 45-50, and 15-20% of an untreated control, respectively), while the alpha-helical content of the enzyme fell exponentially with time. A 7-fold molar excess of Ag+ reduced both the enzymic activity and the alpha-helical content to a much greater degree and no restoration of the enzymic activity was observed. The Km values of Ag+-inactivated enzyme for NADH and oxaloacetate were the same as those of the native enzyme. The data suggest that Ag+ could inhibit enzymic activity both by reducing the structural regularity of the enzyme molecule and by attacking sulfhydryl groups necessary for the catalytic activity of bovine cerebral M-MDH.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.     

Thyroid peroxidase selects the mechanism of either 1- or 2-electron oxidation of phenols, depending on their substituents

Unlike lactoperoxidase and horseradish peroxidase, thyroid peroxidase catalyzed the oxidation of hydroquinone mostly by way of 2-electron transfer. This conclusion could be derived from three independent experiments: ESR measurements of p-benzosemiquinone, trapping the unpaired electron by cytochrome c, and spectrophotometric analysis of catalytic intermediates of the enzymes. The 1-electron flux for hydroquinone oxidation was found to be 15-19% in the reaction of thyroid peroxidase, while it was nearly 100% in the reactions of lactoperoxidase and horseradish peroxidase. From the spectrophotometric analysis of the catalytic intermediates of enzyme, it was suggested that the mechanism of oxidation catalyzed by thyroid peroxidase changes from a 2-electron to a 1-electron type as the substituents at 2- and 6-positions of phenol become bulky or heavy. On the other hand, the mechanism was invariably a 1-electron type when the oxidation of phenols was catalyzed by lactoperoxidase or horseradish peroxidase. These three peroxidases all catalyzed 1-electron oxidation of ascorbate.     

The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.     

First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.     

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.     

Purification and properties of a novel arylmalonate decarboxylase from Alcaligenes bronchisepticus KU 1201.

A novel decarboxylase which catalyzes an enantioselective decarboxylation of alpha-aryl-alpha-methylmalonates to alpha-arylpropionates has been purified from a soil bacterium Alcaligenes bronchisepticus KU 1201. The enzyme was purified 300-fold to homogeneity, judged from the analysis of N-terminal amino acid sequence, and found to be a monomeric enzyme of apparent 24 kDa. The enzyme catalyzes a decarboxylation giving alpha-arylalkanoates from substituted malonates such as alpha-arylmalonate and alpha-alkyl-alpha-arylmalonates. The decarboxylase is not a biotin containing enzyme because avidin have no influence on the enzyme activity. In addition, the enzyme does not require known co-factors (ATP, ADP and coenzyme A) for maximum activity. The enzyme activity was inhibited by sulfhydryl agents. The electronic effect of the substituents on kcat for the enzymic decarboxylation of arylmalonates has been studied. The logarithm of relative value of kcat gave a linear correlation to Hammett's sigma with a rho value of +1.9, for substituted phenylmalonates. Comparing the relative activities, it is clear that the enzyme prefers alpha-arylmalonates to alpha-aryl-alpha-methylmalonates. Thus, the enzyme was tentatively named as arylmalonate decarboxylase.     

Enzymatic lipid peroxidation

Biosynthesis of certain biologically active substances (prostaglandins, thromboxanes, prostacyclins and leukotrienes) in animal tissues occurs with participation of cyclooxygenases and lipoxygenases, enzymic systems of lipid peroxidation. In normal physiological and pathological processes the enzymic lipid peroxidation by microsomal dioxygenases is considerably more active than the nonenzymic one in the same membrane structures. The molecular structure of the products of the enzymic and nonenzymic peroxidation of lipids also differs essentially. An assumption is advanced that cytosol lipoxygenase may be an easily dissociating component of the cyclooxygenase multienzymic complex and its transition from the biomembrane to the cell cytoplasm is accompanied by changes in the enzyme conformation and chemical nature of the products resulted from polyenic lipids oxidation catalyzed by the enzyme.