ABSTRACT
The comparative kinetic study of two glucose oxidizing enzymes, FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase, is presented in the artificial electron transfer mediator system based on ruthenium(III) compounds. It is demonstrated that FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase follow Michaelis kinetics in the D-glucose/ruthenium(III) system. PQQ-dependent glucose dehydrogenase is more active than FAD-dependent glucose oxidase in the process of D-glucose oxidation by ruthenium(III) compounds, this being due to the different catalytic mechanisms of these enzymes.
Subject(s)
Glucose Dehydrogenases/chemistry , Glucose Oxidase/chemistry , Glucose/chemistry , Ruthenium/chemistry , Catalysis , Glucose/metabolism , Glucose Dehydrogenases/metabolism , Glucose Oxidase/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Structure , Oxidation-Reduction/drug effects , Quinolones/chemistry , Quinones/chemistry , Ruthenium/pharmacology , Stereoisomerism , Structure-Activity Relationship , Time FactorsSubject(s)
Glucose Oxidase/chemistry , Organometallic Compounds/chemical synthesis , Peroxidases/chemistry , Ruthenium/chemistry , Aspergillus niger/enzymology , Catalysis , Chemical Phenomena , Chemistry, Physical , Electron Transport , Glucose Oxidase/metabolism , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Molecular Conformation , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Organometallic Compounds/chemistry , Oxidation-Reduction , Peroxidases/metabolism , Spectrophotometry, Infrared , Spectrophotometry, UltravioletABSTRACT
A method for determination of endo-1,4-beta-D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.
Subject(s)
Cellulase/metabolism , Carboxymethylcellulose Sodium , Cellulase/analysis , Cellulase/standards , Electrochemistry/instrumentation , Reference Standards , ViscosityABSTRACT
Glucose oxidase-catalyzed reduction of cis-[MIII(LL)2Cl2]+ (M = Os and Ru) complexes to cis-[MII(LL)2Cl2] (LL = 2,2'-bipyridine and 1,10-phenanthroline type ligands) by D-glucose is a first-order process in the complex and the enzyme in aqueous buffered solution. The reaction follows Michaelis-Menten kinetics in D-glucose and the rate is independent of D-glucose concentration above 0.03 M. The reactivity decreases in the series [Ru(bpy)2Cl2]+ > [Os(phen)2Cl2]+ > [Os(4,4'-Me2bpy)2Cl2]+ > [Os(4,7-Me2phen)2Cl2]+. The measured second-order rate constant for the oxidation of reduced glucose oxidase by [Os(phen)2Cl2]+ in air equals 1.2 x 10(5) M-1 s-1 at pH 6,7, [D-glucose] 0.05 M, and 25 degrees C, which is ca. 20% less than that when the reaction solutions are purged with argon. In the case of [Ru(bpy)2Cl2]+ the rate constant equals 1.8 x 10(5) M-1 s-1 under similar conditions in air, showing higher reactivity of Ru complexes compared with Os ones. The reduction is pH-dependent with a maximum around 7. Added for solubilization of poorly soluble metal complexes, surfactants decrease the rates of the enzymatic reaction. The retardation effect increases in the series: cetyltrimethylammonium bromide < Triton X-100 << sodium dodecyl sulfate, i.e. on going from positively charged to neutral and then to negatively charged surfactants. The behavior of the OsIII and RuIII complexes toward reduced glucose oxidase contrasts to that of recently studied ferricenium cations. As opposed to the latter, the former do not show kinetically meaningful binding with the enzyme, and the Michaelis kinetics typical of the ferricenium case is not realized for the OsIII, and RuIII species. The systems OsIII- or RuIII-glucose oxidase are convenient for routine "one pot" spectrophotometric monitoring of the D-glucose content in samples, since the metal reduction to MII is accompanied by a strong increase in absorbance in the visible spectral region.
Subject(s)
Glucose Oxidase/metabolism , Glucose/metabolism , Osmium Compounds/metabolism , Ruthenium Compounds/metabolism , Aspergillus niger/enzymology , Catalysis , Electron Transport , Hydrogen-Ion Concentration , Kinetics , Models, Chemical , Oxidation-Reduction , Spectrophotometry, AtomicABSTRACT
Reactivity of horseradish peroxidase compounds I and II (HRP-I and HRP-II) toward organometallicic substrates, viz water-soluble ferrocenes RFc (R = COOH and CH2NMe2), has been studied at 25 degrees C, pH 6.0 and ionic strength 0.1 M. The second-order rate constants k2 for the reaction of HRP-I with FcCOOH and FcCH2NMe2 equal (1.00 +/- 0.04) x 10(6) and (0.27 +/- 0.01) x 10(6) M-1 s-1, respectively. The values of k3 for the reaction of HRP-II with FcCOOH and FcCH2NMe2 equal (1.1 +/- 0.1) x 10(4) and (0.25 +/- 0.01) x 10(4) M-1 s-1, respectively. The steady-state kinetic study of the HRP-catalyzed oxidation of the ferrocenes by H2O2 under the same conditions gave the second-order rate constants of (0.94 +/- 0.03) x 10(4) and (0.24 +/- 0.06) x 10(4) M-1 s-1 for FcCOOH and FcCH2NMe2, respectively, which are in a good agreement with k3. The results reported here confirm the proposal that the rate-limiting step of the steady-state oxidation of ferrocenes is the electron transfer from the substrate to HRP-II.
Subject(s)
Horseradish Peroxidase/metabolism , Organometallic Compounds/metabolism , Catalysis , Ferrous Compounds/metabolism , Horseradish Peroxidase/chemistry , Kinetics , Metallocenes , Organometallic Compounds/chemistry , Oxidation-Reduction , Spectrophotometry , Substrate SpecificityABSTRACT
The rate constants for the H2O2-induced irreversible inactivation (kinact) of chloroperoxidase from Caldariomyces fumago evaluated from the analysis of complete kinetic curves of chlorination or bromination of monochlorodimedon were found to follow the rate law kinact = k[H2O2]/(K + [H2O2]) with k = 0.009 > or = 0.002 and 0.0095 > or = 0.010 s-1 and K = (13 > or = 4) x 10(-3) and (9 > or = 2) x 10(-3) M in the presence of 0.01 M chloride and bromide, respectively, at pH 2.75 and 25 degrees C. The data show that chloroperoxidase investigated is more than by a factor of 10 less resistant toward hydrogen peroxide compared to horseradish peroxidase. The possible reason for it and the biotechnological implications are briefly discussed.
Subject(s)
Chloride Peroxidase/metabolism , Hydrogen Peroxide/pharmacology , Mitosporic Fungi/enzymology , Bromides/metabolism , Chlorides/metabolism , KineticsABSTRACT
A novel 'thermodynamic' mechanistic rationale of haloperoxidase catalysis is based on the following two assumptions: (i) the role of enzyme consists only in the rapid equilibration between the halogen-containing species originating from halide and hydrogen peroxide; (ii) the interaction between the enzyme and organic substrate is kinetically insignificant and halogenation occurs as a result of the electrophilic attack of the active brominating (Br3-, Br2 and HBrO) or chlorinating (HCIO) species at monochlorodimedon indicative of a higher chloride 'specificity' of chloroperoxidase from C. fumago.
Subject(s)
Chloride Peroxidase/chemistry , Chloride Peroxidase/metabolism , Peroxidases/chemistry , Peroxidases/metabolism , Catalysis , Kinetics , Mitosporic Fungi/enzymology , Models, Theoretical , Substrate Specificity , ThermodynamicsABSTRACT
The reaction of amylose with NaH and FcCH2NMe3+I- in dimethyl sulfoxide brought about a redox-labeled polymer with a low degree of modification, viz. 1 ferrocene residue per 60 glucose units. The preparation, (0.017ferrocene)amylose, displays one-electron irreversible behavior at a pyrographite electrode in terms of the Delahay formalism, the formal redox potential E degree' being equal to 0.38 V at pH 6 and 40 degrees C versus SCE. Specific to amylose enzymes endo-depolymerases, which carry out random hydrolysis, accept the labeled amylose providing a significant increase in the peak current on cyclic voltammograms. The absence of potential drifts suggests that the effect is due an increase in the diffusion coefficients of the amylose fragments in the course of enzymatic digestion of (0.017ferrocene)amylose. This proposal was confirmed by the simulation of experimental cyclic voltammograms. Several practical applications of the results of this study for electrochemical assaying the amylolytic activity and evaluation of the mechanisms of the enzymatic catalysis by amylases have been demonstrated.
Subject(s)
Amylose/analogs & derivatives , Ferrous Compounds/chemistry , Polymers/chemistry , Amylose/chemical synthesis , Amylose/chemistry , Biodegradation, Environmental , Carbohydrate Sequence , Electrochemistry , Ferrous Compounds/chemical synthesis , Hydrolysis , Metallocenes , Molecular Sequence Data , Oxidation-ReductionABSTRACT
The ferricenium salt FcH+BF4- behaves as a specific substrate of glucose oxidase acting instead of dioxygen with the effective parameters of the Michaelis-Menten equation kcat = 2.60 x 10(2) s-1 and KM = 2.7 x 10(-4) M at 25 degrees C, pH 6.8 and [D-glucose] = 0.001 M. The activity of glucose oxidase can easily be monitored by conventional UV-vis spectrophotometry at 617 nm following the bleaching of the ferricenium dye.
