Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

Catalytic and structural characteristics of carp hepatopancreas D-amino acid oxidase expressed in Escherichia coli

D-amino acid oxidase of carp (Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg(-1) protein toward D-alanine as a substrate. It showed the highest activity toward D-alanine with a low Km of 0.23 mM and a high kcat of 190 s(-1) compared to 10 s(-1) of the pig kidney enzyme. Nonpolar and polar uncharged D-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 alpha-helices and 17 beta-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney D-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp D-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities.     

Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.     

D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.     

Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica

Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signalling molecules in plants under abiotic and biotic stresses. Calluses from Populus euphratica, which show salt tolerance, were used to study the interaction of NO and H2O2 in plant adaptation to salt resistance. The nitric oxide synthase (NOS) activity was identified in the calluses, and this activity was induced under 150 mM NaCl treatment. Under 150 mM NaCl treatment, the sodium (Na) percentage decreased, but the potassium (K) percentage and the K/Na ratio increased in P. euphratica calluses. Application of glucose/glucose oxidase (G/GO, a H2O2 donor) and sodium nitroprusside (SNP, a NO donor) revealed that both H2O2 and NO resulted in increased K/Na ratio in a concentration-dependent manner. Diphenylene iodonium (DPI, an NADPH oxidase inhibitor) counteracted H2O2 and NO effect by increasing the Na percentage, decreasing the K percentage and K/Na ratio. NG-monomethyl-L-Arg monoacetate (NMMA, an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO, a specific NO scavenger) only reversed NO effect, but did not block H2O2 effect. The increased activity of plasma membrane (PM) H+ -ATPase caused by salt stress was reversed by treatment with DPI and NMMA. Exogenous H2O2 increased the activity of PM H+ -ATPase, but the effect could not be diminished by NMMA and PTIO. The NO-induced increase of PM H+ -ATPase can be reversed by NMMA and PTIO, but not by DPI. Western blot analysis demonstrated that NO and H2O2 stimulated the expression of PM H+ -ATPase in P. euphratica calluses. These results indicate that NO and H2O2 served as intermediate molecules in inducing salt resistance in the calluses from P. euphratica under slat stress by increasing the K/Na ratio, which was dependent on the increased PM H+ -ATPase activity.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Interactions of hydrogen peroxide with ribulose bisphosphate carboxylase oxygenase

Hydrogen peroxide inhibited both carboxylase and oxygenase activities of purified, and fully activated, spinach ribulose-1,5-bisphosphate (RuP2) carboxylase-oxygenase. Inhibition of the carboxylase reaction was mixed competitive with respect to CO2 (Ki = 1.2 mM) and uncompetitive with respect to RuP2. For the oxygenase reaction, H2O2 was a competitive inhibitor with respect to O2 (Ki = 2.1 mM) and an uncompetitive inhibitor with respect to RuP2. H2O2 did not alter the stoichiometry between CO2 and RuP2 in the carboxylase reaction, indicating that H2O2 was not itself a substrate for the enzyme. RuP2 decreased the rate of deactivation of the enzyme which occurred at limiting CO2 concentrations. H2O2 greatly enhanced this stabilizing effect of RuP2 but had no effect on the rate of deactivation in the absence of RuP2. The inhibitory and stabilizing effects of H2O2 varied similarly with H2O2 concentration. These instantaneous, reversible effects of H2O2 were readily distinguishable from an irreversible inhibitory effect which occurred quite slowly, and in the absence of RuP2. These observations are discussed in relation to the enzyme's catalytic mechanism and its activation-deactivation transformations.     

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.     

Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H(2)O(2) Production by Pleurotus eryngii

The existence of a redox cycle leading to the production of hydrogen peroxide (H(2)O(2)) in the white rot fungus Pleurotus eryngii has been confirmed by incubations of 10-day-old mycelium with veratryl (3,4-dimethoxybenzyl) and anisyl (4-methoxybenzyl) compounds (alcohols, aldehydes, and acids). Veratraldehyde and anisaldehyde were reduced by aryl-alcohol dehydrogenase to their corresponding alcohols, which were oxidized by aryl-alcohol oxidase, producing H(2)O(2). Veratric and anisic acids were incorporated into the cycle after their reduction, which was catalyzed by aryl-aldehyde dehydrogenase. With the use of different initial concentrations of either veratryl alcohol, veratraldehyde, or veratric acid (0.5 to 4.0 mM), around 94% of veratraldehyde and 3% of veratryl alcohol (compared with initial concentrations) and trace amounts of veratric acid were found when equilibrium between reductive and oxidative activities had been reached, regardless of the initial compound used. At concentrations higher than 1 mM, veratric acid was not transformed, and at 1.0 mM, it produced a negative effect on the activities of aryl-alcohol oxidase and both dehydrogenases. H(2)O(2) levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H(2)O(2)-reducing system kept these levels steady. On the other hand, the concomitant production of the three above-mentioned enzymes during the active growth phase of the fungus was demonstrated. Finally, the possibility that anisaldehyde is the metabolite produced by P. eryngii for the maintenance of this redox cycle is discussed.     

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.     

Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide

The lysosomotropic amines methylamine (40 mM) and chloroquine (125 mM) prevented the killing of cultured hepatocytes by hydrogen peroxide generated in the medium by glucose oxidase. Maximum protection required several hours preincubation with either amine. Sensitivity of the hepatocytes to H2O2 was restored either by the addition of ferrous or ferric iron to the culture medium, or by incubating the cells for 4 hours in the absence of either amine prior to treatment with H2O2. Neither methylamine nor chloroquine had any effect on the cell killing by t-butyl hydroperoxide, a hepatotoxin that does not require iron. The protective effect of the lysosomotropic amines was distinguished from that of the ferric iron chelator deferoxamine in two ways: 1) deferoxamine protected hepatocytes from H2O2 toxicity but did not require a pretreatment period; and 2) in contrast to methylamine or chloroquine, deferoxamine had no effect on lysosomal pH as assessed by the fluorescent probe acridine orange. The data suggest that a lysosomal pool is the source of the ferric iron necessary for the killing of hepatocytes by H2O2.     

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.     

pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results.