Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.     

Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. strain AK-5

Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.     

Bioorganic mechanisms of the formation of free radicals catalyzed by glucose oxidase

In this communication, we have described the activation of several xenobiotics by glucose oxidase from Aspergillus niger. The following compounds are readily reduced by d-glucose, in the presence of glucose oxidase: p-nitroso-N,N-dimethylaniline, methyl-1,4-benzoquinone, and 7,7,8,8-tetracyano-quinodimethane. In each case, the products of enzymatic reduction undergo a dismutation reaction with the parent compound and thus afford the formation of free radicals. In some cases, and at an appropriate pH value, the transformation of a parent compound into free radicals is almost quantitative. Under optimal conditions, free radicals are stable for several minutes in aqueous solutions under physiological conditions.     

Study of the protein-ubiquinone interaction in succinate-cytochrome C reductase with azido-ubiquinone derivatives

Several azido-ubiquinones have been synthesized for the study of protein-ubiquinone interaction in succinate-cytochrome $\text{c}$ reductase. In the absence of light, azido-ubiquinones are partially effective in restoring enzymatic activity to ubiquinone- and phospholipid-depleted reductase and the binding of azido-ubiquinones can be partially reversed by 5-(10-bromodecyl)-ubiquinone. When 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone reactivated reductase is illuminated with long wavelength UV light, a complete and irreversible inhibition is observed. This specific photo-inactivation, exerted only by 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone, and not by other azido-ubiquinone derivatives, is evidence for the existence of a specific benzoquinone ring binding site in the enzyme.     

Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles

A sensitive enzymatic assay for oxidase reactions both in aqueous solution and in hexadecyltrimethylammoniumbromide (CTAB) reversed micelles has been developed. The assay is based on the fluorescence detection of dichlorofluorescein, which is formed by hydrogen peroxide oxidation of the nonfluorescent precursor dichlorofluorescin. Hydrogen peroxide as product of the reaction catalyzed by glucose oxidase served to select the reaction conditions. The reaction rate is distinctly enhanced in CTAB reversed micelles as compared to the rate in aqueous solution. This effect, combined with the high sensitivity owing to the strong fluorescence of dichlorofluorescein, makes the assay attractive for the detection of low enzyme, substrate, or peroxide concentrations.     

Purification and characterization of an intracellular NADH: quinone reductase from Trametes versicolor

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41 kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between 20-40 degrees C , with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by CuSO(4), HgCl(2), MgSO(4), MnSO(4), AgNO(3), dicumarol, KCN, NaN(3), and EDTA. Its Km and Vmax with NADH as an electron donor were 23 microM and 101 mM/mg per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.     

Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay

An online immobilized glucose oxidase (GOx) capillary microreactor was developed based on an enzymatic redox reaction with 1,4-benzoquinone as an acceptor of electrons, replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation. A high efficiency of enzymolysis was obtained at 1 mg ml^?1 1,4-benzoquinone for 5 min of incubation at 25 °C, and baseline separation of the substrate and product could be achieved with a resolution of 3.85 by employing 20 mM phosphate buffer (pH 8.0) containing 40 mg ml^?1 sulfated β-cyclodextrin as an additive, a constant voltage of 15 kV, and a detection wavelength of 220 nm. In addition, an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag⁺ and Cu²⁺ used as model inhibitors. The results indicate that Ag⁺ (IC₅₀ = 69.16 μM) has a markedly higher inhibitory effect than Cu²⁺ (IC₅₀ = 1.33 mM). The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of beta-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/K(B), for reactions of these substrates were collected from pH 2.5-8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of beta-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid. Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Thymoquinone,a biologically active component of Nigella sativa,induces mitochondrial production of reactive oxygen species and programmed death of tumor cells

Biophysics - Mechanisms of tumor-cell responses to 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and 1,4-benzoquinone were studied using fluorescence and the inhibition assay. It was shown...     

Bioluminescence characteristics of Lake Shira water

Bioluminescence bioassays based on luminous bacteria (Photobacterium phosphopreum) and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. The enzymatic system in the presence of 1,4-benzoquinone are shown to be more sensitive to redox characteristics of the salt water than this in the absence of 1,4-benzoquinone. 1,4-benzoquinone should be applied for the preparation of a model solution for the monitoring of redox properties of the salt water. Using this technique, the results of bioluminescence analysis are used to construct a heterogeneity map that characterizes the spatial and temporal water quality of lake Shira. A partial map was based on the bioluminescence characteristics of water samples taken along the shoreline, sampling stations in the different places and in different depths of the lake. It has been demonstrated that the bioluminescence assay measurements must be done within two hours after the sampling time.     

CLK-1/Coq7p is a DMQ mono-oxygenase and a new member of the di-iron carboxylate protein family.

Strains of Caenorhabditis elegans mutant for clk-1 exhibit a 20-40% increase in mean lifespan. clk-1 encodes a mitochondrial protein thought to be either an enzyme or regulatory molecule acting within the ubiquinone biosynthesis pathway. Here CLK-1 is shown to be related to the ubiquinol oxidase, alternative oxidase, and belong to the functionally diverse di-iron-carboxylate protein family which includes bacterioferritin and methane mono-oxygenase. Construction and analysis of a homology model indicates CLK-1 is a 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone mono-oxygenase as originally predicted. Analysis of known CLK-1/Coq7p mutations also supports this notion. These findings raise the possibility of developing CLK-1-specific inhibitors to test for lifespan extension in higher organisms.     

Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone

Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms.     

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.     

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.     

Synthesis and purification of 3-hydroxykynurenine-O-beta-glucoside, a primate lens ultraviolet filter, and its application in a two-step assay for beta-glucosidase activity.

3-Hydroxykynurenine-3-O-beta-glucoside (3-HKG) functions in the primate lens as a filter of 295- 400-nm light, thereby protecting the retina from damaging UV radiation. Although extensive studies have been conducted to determine the functional role of 3-HKG in the primate lens, an efficient method for its synthesis and purification has yet to be developed. Several procedures have been reported for the synthesis of 3-HKG; however, these procedures either result in low yields or require numerous sequential reactions and purification steps. In this study, we report a two-step synthesis of 3-HKG with a one-step purification and a two- to eightfold increase in yield over previously reported methods. Additionally, an assay was developed to confirm the presence of a beta-glycosidic linkage in the purified reaction product and we propose a method by which 3-HKG can be used as a general probe of beta-glucosidase activity. The assay consists of adding glucose oxidase to the 3-HKG/glucosidase solution and then allowing the hydrogen peroxide, generated from the interaction of glucose with glucose oxidase, to oxidize 3-hydroxykynurenine to xanthomattin (XAN) and 4,6-dihydroxyquinolinequinone carboxylic acid (DHQCA). Both XAN and DHQCA absorb strongly between 400 and 500 nm and the color change of the solution can be seen by eye. In addition, XAN fluoresces in the visible region with lambda(max) = 527 nm.     

Characterization of pyranose oxidase variants for bioelectrocatalytic applications

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H₂O₂. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (I_max^app/K_M^app) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.