Purification of two peroxidase isoenzymes of Aloe barbadensis which oxidize p-coumaric acid

Using a combination of hydrophobicity and ion-exchange chromatography methods, one cationic (pI 9.0) and one anionic (pI 4.5) peroxidase (donor: hydrogen-peroxide oxidoreductase; EC 1.11.1.7) isoenzymes of Aloe barbadensis have been purified (the cationic peroxidase to homogeneity as judged by SDS-PAGE analysis and microsequencing). This allowed us to initiate the investigation of individual catalytic properties to be related to their respective functions in vivo. The two peroxidases have an optimal activity at pH 6.0. Apparent affinities for H₂O₂ range between 0.01 and 0.14 mM depending on the phenolic substrate and the isoenzyme. The apparent K_m values for the phenolics (p-coumaric acid and hydroquinone) are some 25-fold lower in the anionic (around 0.02 mM) than in the cationic (around 0.77 and 0.34 mM, respectively) isoenzyme. The possible functions of the activities are discussed.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K_Mox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK_Mox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K_Mox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K_Mox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K_Mox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.     

Reaction mechanism of grape catechol oxidase—a kinetic study

The initial velocity of the oxidation of 4-methylcatechol by grape catechol oxidase was determined. The kinetic analysis indicates that first there is random binding of an oxygen and a 4-methylcatechol molecule to the enzyme. Then one product molecule is released prior to the binding of second 4-methylcatechol molecule which is followed by the release of a second product molecule. The true K_m values were determined; they were found to be 0.5 mM for oxygen and 17 mM for 4-methylcatechol.     

Effect of different phenols on the nadh-oxidation catalysed by a peroxidase from lupin

Cell wall-bound peroxidase (EC 1.11.1.7) from lupin (Lupinus albus) shows a transition from oxidase to peroxidase activity when it oxidizes NADH. The oxidase phase represents a lag period in the time course of the reaction. This phase is phenol-dependent and responsible for hydrogen peroxide formation. Guaiacol, an assay substrate, and p-coumaric, ferulic and sinapic acids, precursors of the cinnamyl alcohols used in the lignification process affect both the length of lag period and the rate of the peroxidase phase of NADH oxidation. The effect of different phenols on the time course of the reaction is related to the efficacy (V_max/K_m ratio) of the enzyme when it is acting on them as a peroxidese.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Properties of hydroxycinnamate: CoA ligase from stems of Salix babylonica cultured in vitro

Hydroxycinnamate: CoA ligase was extracted from stems of in vitro willow cultures and characterized. One peak of activity was obtained after column chromatography on Sephadex G 100 or DEAE Sephacel. p-Coumaric acid gave the highest V_max among the cinnamates examined. The K_mvalues for p-coumaric, caffeic and ferulic acid were 31.0, 4.7 and 46 μM, respectively. The MW of the CoA ligase was 57 000 and the pH optimum was 7.0. The characteristics of the enzyme correspond to its physiological role in lignin biosynthesis.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

Soybean root growth inhibition and lignification induced by p-coumaric acid

The effects of 0.25–2 mM p-coumaric acid, a phenylpropanoid metabolite with recognized allelopathic properties, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) activities, soluble and cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H₂O₂) level and lignin content and its monomeric composition in soybean (Glycine max (L.) Merr.) roots. At ≥0.25 mM, exogenously supplied p-coumaric acid induced premature cessation of root growth, increased POD activity and lignin content and decreased the H₂O₂ content. At ≥0.5 mM, the allelochemical decreased the cell viability and PAL activity. When applied jointly with PIP (an inhibitor of the cinnamate 4-hydroxylase, C4H), 1 mM p-coumaric acid increased lignin content. In contrast, the application of MDCA (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with p-coumaric acid did not increase lignin content. The lignin monomeric composition of p-coumaric acid-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and guaiacyl (G) units. Taken together, these results suggest that p-coumaric acid's mode of action is entry via the phenylpropanoid pathway, resulting in an increase of H and G lignin monomers that solidify the cell wall and restrict soybean root growth.     

O-Methylation of flavonoid substrates by a partially purified enzyme from soybean cell suspension cultures.

A methyltransferase, which catalyzes the methylation of luteolin (K_m, 16 μM) using S-adenosyl-l-methionine as the methyl donor, has been purified about 38-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The following 3,4-dihydroxy phenolic compounds were also methylated: luteolin 7-O-glucoside (K_m, 28 μm), quercetin (K_m, 35 μm), eriodictyol (K_m, 75 μm), 5-hydroxyferulic acid (K_m, 227 μm), dihydroquercetin (K_m, 435 μm), and caffeic acid (K_m, 770 μm). Rutin and quercetin 3-O-glucoside were poor substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of p-coumaric acid, m-coumaric acid, ferulic acid, isoferulic acid, sinapic acid, apigenin, or naringenin. While the isoflavones biochanin A and daidzein did not serve as substrates, texasin (6,7-dihydroxy-3′-methoxyisoflavone) was methylated (K_m, 35 μm). The methylation of caffeic acid and quercetin showed a pH optimum of 8.6–8.9. The enzyme required Mg²⁺ ions for maximum activity (approximately 1 mm) and could be totally inhibited by EDTA (10 mm). The K_m for S-adenosyl-l-methionine was 11 μm. S-Adenosyl-l-homocysteine inhibited the methylation of luteolin by S-adenosyl-l-methionine.     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.     

Grape skins (Vitis vinifera L.) catalyze the in?vitro enzymatic hydroxylation of p-coumaric acid to caffeic acid

The ability of grape skins to catalyze in vitro conversion of p-coumaric acid to the more potent antioxidant caffeic acid was studied. Addition of different concentrations of p-coumaric to red grape skins (Cabernet Sauvignon) resulted in formation of caffeic acid. This caffeic acid formation (Y) correlated positively and linearly to p-coumaric acid consumption (X): Y = 0.5 X + 9.5; R ² = 0.96, P < 0.0001. The kinetics of caffeic acid formation with time in response to initial p-coumaric acid levels and at different grape skin concentrations, indicated that the grape skins harboured an o-hydroxylation activity, proposedly a monophenol- or a flavonoid 3′-monooxygenase activity (EC 1.14.18.1 or EC 1.14.13.21). The K _m of this crude o-hydroxylation activity in the red grape skin was 0.5 mM with p-coumaric acid.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii

A formaldehyde oxidase activity was found in cell-free extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 4₈, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The K _m value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g.2. NADH, succinate, d(?)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(?)- and l(+)-lactate dehydrogenases were NAD⁺ and NADP⁺ independent. Ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases.3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(?)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(?)-lactate and l(+)-lactate oxidases.4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation.5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(?)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide.6. The apparent K_m for O₂ for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K_m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O₂.     

Hepatic microsomal ethanol-oxidizing system: solubilization, isolation, and characterization

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD⁺ to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K_m for ethanol (7.2 mm, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 mm) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K_m for oxygen (8.3 μm), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H₂O₂-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.     

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; K_m, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl₂, p-chloromercuribenzoic acid, and CuCl₂ inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53°C to 58°C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol. m⁻¹ mg⁻¹ protein and an apparent K_m for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.