Pulse radiolysis studies on galactose oxidase

Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu(II), no Tyr(*) radical) reacts with pulse radiolysis generated formate radicals CO(2)(*-) to give an intermediate UV-vis spectrum assigned as RSSR(*-), peak at 450 nm (epsilon = 8100 M(-1) cm(-1)). From a detailed kinetic analysis at 450 nm, pH 7.0, the following steps have been identified. First the strongly reducing CO(2)(*-) (-1.9V) reduces GOase(semi) (k(0) > or = 6.5 x 10(8) M(-1) s(-1)) to a species GOase(semi)(*-). This is followed by biphasic reactions (i) GOase(semi)(*-) + GOase(semi) (k(1) = 1.6 x 10(7) M(-1) s(-1)) to give GOase(semi) + P(*-) and (ii) P(*-) + GOase(semi) (k(2) = 6.7 x 10(6) M(-1) s(-1)) to give GOase(semi)RSSR(*-). There are no significant absorbance changes for the formation of GOase(semi)(*-) and P(*-), which are Cu(I) (or related) species. However, GOase(semi)RSSR(*-) has an absorption spectrum which differs significantly from that of GOase(semi). The 450 nm peak is characteristic of an RSSR(*-) radical with two cysteines in close sequence proximity and is here assigned to Cys515-Cys518, which is at the GOase surface and 10.2 A from the Cu. On chemical modification of the RSSR group with HSPO(3)(2-) to give RSSPO(3)H(-) and RS(-), absorbance changes are approximately 50% of those previously observed. The decay of RSSR(*-) (0.17 s(-1)) results in the formation of GOase(red). No RSSR(*-) formation is observed in the reaction of GOase(semi) Tyr495Phe with CO(2)(*-), and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(ox) with CO(2)(*-), a single-stage reaction gives GOase(semi).     

Bioelectrochemical response of the polyaniline galactose oxidase electrode

Galactose oxidase has been immobilized in a polyaniline film. The response current of the galactose oxidase electrode is a function of the applied potential and increases as the pH increases from 5.61 to 7.25. The optimum pH of the immobilized galactose oxidase is 7.25. The activation energy of the enzyme-catalysed reaction is 41.8 kJ mol⁻¹. The response current of the enzyme electrode shows good reproducibility at temperatures below the optimum temperature of 30.4°C and increases as the galactose concentration increases from 0.2 to 6 mmol dm⁻³. Thus the polyaniline galactose oxidase electrode can be used to determine galactose concentration.     

Heavily fluorinated carbohydrates as enzyme substrates: oxidation of tetrafluorinated galactose by galactose oxidase

Galactose oxidase (GOase) was shown to oxidise several C2/C3 fluorinated galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated sugar.     

Galactosyl-biomimetic dye-ligands for the purification of Dactylium dendroides galactose oxidase

Two anthraquinone galactosyl-biomimetic dye-ligands comprising, as terminal biomimetic moiety, galactose analogues (1-amino-1-deoxy-beta-D-galactose and D(+)-galactosamine) were designed for the enzyme galactose oxidase (GAO), using molecular modelling, synthesized and characterized. The biomimetic ligands were immobilized on agarose beads and the affinity adsorbents, together with a non-biomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their ability to purify GAO from Dactylium dendroides. Both biomimetic adsorbents showed higher purifying ability for GAO compared to the non-biomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising, as terminal biomimetic moiety, 1-amino-1-deoxy-beta-D-galactose (BM1) exhibited the highest purifying ability for GAO. This affinity adsorbent did not bind galactose dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, or glucose oxidase. The dissociation constant (K(D)) of the immobilized BM1 ligand with GAO was found to be equal to 45.8 microM, whereas the binding capacity was equal to 709 U per ml adsorbent. Therefore, the BMI adsorbent was integrated in a facile two-step purification procedure for GAO. The purified enzyme showed a specific activity equal to 2038 U/mg, the highest reported so far, approximately 74% overall recovery and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis.     

Expression and characterization of a recombinant Fusarium spp. galactose oxidase

The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation. Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.     

Bio-mimicking galactose oxidase and hemocyanin, two dioxygen-processing copper proteins

The modelling of the active sites of metalloproteins is one of the most challenging tasks in bio-inorganic chemistry. Copper proteins form part of this stimulating field of research as copper enzymes are mainly involved in oxidation bio-reactions. Thus, the understanding of the structure-function relationship of their active sites will allow the design of effective and environmental friendly oxidation catalysts. This perspective illustrates some outstanding structural and functional synthetic models of the active site of copper proteins, with special attention given to models of galactose oxidase and hemocyanin.     

Colorimetric quantification of galactose using a nanostructured multi-catalyst system entrapping galactose oxidase and magnetic nanoparticles as peroxidase mimetics

A colorimetric method for quantification of galactose, which utilizes a nanostructured multi-catalyst system consisting of Fe(3)O(4) magnetic nanoparticles (MNPs) and galactose oxidase (Gal Ox) simultaneously entrapped in large pore sized mesocellular silica, is described. Gal Ox, immobilized in a silica matrix, promotes reaction of galactose to generate H(2)O(2) that subsequently activates MNPs in silica mesopores to convert a colorimetric substrate into a colored product. By using this colorimetric method, galactose can be specifically detected. Along with excellent reusability via application of simple magnetic capturing, enhanced operational stability was achieved by employing a cross-linked enzyme aggregate (CLEA) method for Gal Ox immobilization. This protocol leads to effective prevention of enzyme leaching from the pores of mesocellular silica. The analytical utility of the new colorimetric biosensor was demonstrated by its use in diagnosing galactosemia, a genetic metabolic disorder characterized by the inability to utilize galactose, through analysis of clinical dried blood spot specimens. A microscale well-plate format was employed that possesses a multiplexing capability. The multi-catalyst system entrapping Gal Ox and MNPs represents a new approach for rapid, convenient, and cost-effective quantification of galactose in human blood and it holds promise as an alternative method for galactosemia diagnosis, replacing the laborious procedures that are currently in use.     

Cu(II)-nitroxyl radicals as catalytic galactose oxidase mimics

Results from Hammett correlation studies and primary kinetic isotope effects for the CuCl-TEMPO catalysed aerobic benzyl alcohol oxidations are inconsistent with an oxoammonium based mechanism. We postulate a copper-mediated dehydrogenation mechanism, in which TEMPO regenerates the active Cu(II)-species. This mechanism is analogous to that observed for Galactose Oxidase and mimics thereof.     

Catalytic activity of glucose oxidase after dielectrophoretic immobilization on nanoelectrodes

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, for example, antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability of the immobilization method for sensing or research in general, we want to test it for other enzymes, too. In this study, glucose oxidase (GOX) from Aspergillus niger was immobilized on TiN nanoelectrode arrays by DEP. Fluorescence microscopy showed the intrinsic fluorescence of the immobilized enzymes flavin cofactor on the electrodes. The catalytic activity of immobilized GOX was detectable, but a fraction of less than 1.3% of the maximum activity that was expected for a full monolayer of immobilized enzymes on all electrodes was stable for multiple measurement cycles. Therefore, the effect of DEP immobilization on the catalytic activity strongly depends on the used enzyme.     

Glycoprotein labeling using engineered variants of galactose oxidase obtained by directed evolution

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.     

Electrochemical and spectroscopic effects of mixed substituents in bis(phenolate)-copper(II) galactose oxidase model complexes

Nonsymmetric substitution of salen (1(R(1),R(2))) and reduced salen (2(R(1),R(2))) Cu(II)-phenoxyl complexes with a combination of -(t)Bu, -S(i)Pr, and -OMe substituents leads to dramatic differences in their redox and spectroscopic properties, providing insight into the influence of the cysteine-modified tyrosine cofactor in the enzyme galactose oxidase (GO). Using a modified Marcus-Hush analysis, the oxidized copper complexes are characterized as Class II mixed-valent due to the electronic differentiation between the two substituted phenolates. Sulfur K-edge X-ray absorption spectroscopy (XAS) assesses the degree of radical delocalization onto the single sulfur atom of nonsymmetric [1((t)Bu,SMe)](+) at 7%, consistent with other spectroscopic and electrochemical results that suggest preferential oxidation of the -SMe bearing phenolate. Estimates of the thermodynamic free-energy difference between the two localized states (ΔG(o)) and reorganizational energies (λ(R(1)R(2))) of [1(R(1),R(2))](+) and [2(R(1),R(2))](+) lead to accurate predictions of the spectroscopically observed IVCT transition energies. Application of the modified Marcus-Hush analysis to GO using parameters determined for [2(R(1),R(2))](+) predicts a ν(max) of ～13600 cm(-1), well within the energy range of the broad Vis-NIR band displayed by the enzyme.     

Intramolecular charge transfer and biomimetic reaction kinetics in galactose oxidase model complexes

One-electron oxidation of two structurally similar CuII-diphenolate complexes, 1 and 2, creates EPR-silent CuII-phenoxyl complexes [1]+ and [2]+ that mimic the oxidized form of the enzyme galactose oxidase (GOase). Both model complexes display novel NIR absorptions assigned to phenolate-phenoxyl charge transfer that resemble a tyrosinate-tyrosyl charge-transfer band observed in the enzymatic system. [1]+ and [2]+ react with benzyl alcohol to form 0.5 equivs of benzaldehyde per complex; biomimetic reduction to CuI-phenol complexes is not observed, but such species may exist transiently. Initial kinetic studies show that [2]+ reacts faster with benzyl alcohol than does [1]+, despite being a significantly weaker oxidant (DeltaE degrees = 370 mV). This acceleration is ascribed to mechanistic differences: [2]+ appears to bind substrate prior to the rate-determining step. Large, nonclassical kinetic isotope effects confirm C-H bond cleavage as the rate-determining step in the reactions of both [1]+ and [2]+ with benzyl alcohol, as is the case for GOase.     

Role of the Tyr-Cys cross-link to the active site properties of galactose oxidase

The catalytically relevant, oxidized state of the active site [Cu(II)-Y·-C] of galactose oxidase (GO) is composed of antiferromagnetically coupled Cu(II) and a post-translationally generated Tyr-Cys radical cofactor [Y·-C]. The thioether bond of the Tyr-Cys cross-link has been shown experimentally to affect the stability, the reduction potential, and the catalytic efficiency of the GO active site. However, the origin of these structural and energetic effects on the GO active site has not yet been investigated in detail. Here we present copper and sulfur K-edge X-ray absorption data and a systematic computational approach for evaluating the role of the Tyr-Cys cross-link in GO. The sulfur contribution of the Tyr-Cys cross-link to the redox active orbital is estimated from sulfur K-edge X-ray absorption spectra of oxidized GO to be about 24 ± 3%, compared to the values from computational models of apo-GO (15%) and holo-GO (22%). The results for the apo-GO computational models are in good agreement with the previously reported value for apo-GO (20 ± 3% from EPR). Surprisingly, the Tyr-Cys cross-link has only a minimal effect on the inner sphere, coordination geometry of the Cu site in the holo-protein. Its effect on the electronic structure is more striking as it facilitates the delocalization of the redox active orbital onto the thioether sulfur derived from Cys, thereby reducing the spin coupling between the [Y·-C] radical and the Cu(II) center (752 cm(-1)) relative to the unsubstituted [Y·] radical and the Cu(II) center (2210 cm(-1)). Energetically, the Tyr-Cys cross-link lowers the reduction potential by about 75 mV (calculated) allowing a more facile oxidation of the holo active site versus the site without the cross-link. Overall, the Tyr-Cys cross-link confers unique ground state properties on the GO active site that tunes its function in a remarkably nuanced fashion.     

Oxidatively robust monophenolate-copper(II) complexes as potential models of galactose oxidase

Cupric complexes of a novel phenanthroline-phenolate ligand have strongly distorted coordination geometries and electrochemical properties conducive to modeling the spectroscopy and reactivity of the enzyme galactose oxidase.     

In situ preparation of beta-D-1-O-hydroxylamino carbohydrate polymers mediated by galactose oxidase

[reaction: see text] Galactose oxidase produced a C-6 aldehyde in various terminal-containing galactose hydroxylamines for the simultaneous in situ generation of an A-B type condensation for the construction of unique oxime polymers. Molecular weights of the corresponding polymers were determined to be in the range of 4200-8900 g/mol, respectively. This indicates that approximately 20-25 sugar units were incorporated in these unique polymers.     

N-Salicylideneaminoacidato copper(II) complexes as galactose oxidase model compounds

[CuL·B]^q model systems, where L²⁻ is the tridentate Schiff base ligand formed by the condensation of salicylaldehyde with alanine, B is imidazole, q=−1, 0 and +1, are optimized at B3LYP/6-31G* level of theory. Their electronic structure is described in terms of Mulliken population analysis and reactivity indices of Fukui. The total energy of [CuL·B]^q species increases with the electron removal. The reactivity indices suitable for the alcohol (sugar) adducts formation (CuO_sugar and O_phenoxylH_sugar interactions) are in the neutral molecule as well as in the singlet cation. Despite the similar trends in Cu–O_phenoxyl bonding and significant O_phenoxyl spin density in triplet cation, the catalytic mechanism of sugars oxidation proposed for the galactose oxidase cannot be used in our system because the [CuL·B]⁺ formation is energetically unfavorable. The imidazole nitrogen deprotonation is more probable than of the alanine ternary carbon atom.     

Dissecting an enzyme—model compounds for the galactose oxidase radical site

The active site of the enzyme galactose oxidase (GOase) contains square‐pyramidal monocopper site, one of whose ligands is a tyrosinate side‐chain that is oxidized to an unusually stable radical in the active enzyme. The structure of this non‐innocent tyrosinate is unique in two ways. First, the tyrosine ring is crosslinked to a neighboring cysteine residue, affording an orthoalkylsulfanyl‐substituted phenoxide ligand. Second, this assembly is protected by a π–π interaction to a tryptophan indole group. We describe here a series of compounds designed to model various aspects of the structure of this unusual cofactor. Our studies have shown that the thermodynamic stability of the GOase radical can be attributed almost exclusively to its thioether substituent, that the π–π interaction contributes little to this stability, and that the assignment of the optical spectrum of the GOase radical is more complex than had been previously suggested. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:494–500, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10091     

Structure of the oxidized active site of galactose oxidase from realistic in silico models

A systematic in silico approach is employed to generate an accurate model for the catalytically important oxidized state of galactose oxidase (GO) using spectroscopically calibrated hybrid density-functional theory. GO displays three distinct oxidation states: oxidized [Cu(II)-Y*], semireduced [Cu(II)-Y], and fully reduced [Cu(I)-Y], but only the [Cu(II)-Y*] and the [Cu(I)-Y] states are assumed to be involved in catalysis. We have developed multiple models for the oxidized [Cu(II)-Y*] state, whose structure has not yet been fully characterized. These models were evaluated by comparison of calculated and experimental structural data, singlet-triplet energy gaps, and electronic transitions for the antiferromagnetically coupled oxidized [Cu(II)-Y*] state. An extended model system that includes explicit solvent molecules and second coordination sphere residues (R330, Y405, and W290) is essential to obtain the correct electronic structure of the active site. The model with all the residues that have been shown to affect the radical stability and catalysis resulted in a singlet ground state with the radical centered on the Y272-C228 cofactor. The optimized structure of the oxidized GO [Cu(II)-Y*] reveals a five-coordinated square pyramidal coordination geometry very similar to [Cu(II)-Y] with considerably different Cu-ligand distances. The hydrogen-bonding interactions involving Y495 modulates the spin density distribution and the singlet-triplet energy gaps. The final model as the most reasonable structure of the oxidized [Cu(II)-Y*] state in GO reproduces the spectroscopic signature of oxidized GO.