Galactose Oxidase

McPherson, Michael J.; Parsons, Mark R.; Spooner, R.K.; Wilmot, Carrie M.

doi:10.1002/0470028637.met193

Cited by 4 publications

(5 citation statements)

References 56 publications

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“…The rate of change of absorbance at 415 nm (ε 415 = 31300 M −1 cm −1 ) was monitored on a Shimadzu UV-2401 PC spectrophotometer. One unit of activity is defined as the amount of enzyme required to form 1 μmol of H 2 O 2 /min .…”

Section: Methodsmentioning

confidence: 99%

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

et al. 2008

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Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redoxactive cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (λ max ) 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required. † This work was supported by the Biotechnology and Biological Sciences Research Council (24/B11662) and NIH (GM 27659). S.J. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 October 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe range of chemistry available for enzyme catalysis may be extended by post-translational modifications of encoded amino acids, generating new intrinsic cofactor moieties (1). One general class of post-translationally modified cofactor involves protein-derived radicals, including tyrosyl, glycyl, thiyl, and tryptophanyl radicals (2-5). The second class includes proteins that undergo more extensive post-translational modifications derived by covalent modification of an amino acid, or involving peptide cleavage and modification, or new bondforming reactions. Post-translational modification occurs either by a self-processing pathway or via the involvement of accessory enzymes, and both modes of modification may be utilized by an enzyme to facilitate new reactivity and/or provide additional structural functionality.The most frequently modified residue appears to be tyrosine, which is transformed into a variety of novel structures, including cross-links and qui...

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Section: Methodsmentioning

confidence: 99%

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

et al. 2008

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“…Purity and molecular weight were confirmed by SDS−PAGE. Wild-type and W290F galactose oxidase were alternatively isolated via a Pichia pastoris expression system ( , ). Galactose oxidase purified from the yeast expression system had kinetic and structural properties essentially indistinguishable from those of the enzyme isolated from the fungal expression system.…”

Section: Methodsmentioning

confidence: 99%

“…The absorbance change at 415 nm was recorded on an HP8453 spectrophotometer at 25 °C. Specific activity was calculated from the rate of production of the ABTS radical cation ( 415 ) 31 300 M -1 cm -1 ) assuming two molecules of the ABTS radical cation are produced from every molecule of hydrogen peroxide (48). One unit of specific activity of galactose oxidase corresponds to 1 µmol of H 2 O 2 produced min -1 mg -1 .…”

Section: Methodsmentioning

confidence: 99%

The Stacking Tryptophan of Galactose Oxidase: A Second-Coordination Sphere Residue that Has Profound Effects on Tyrosyl Radical Behavior and Enzyme Catalysis^,

et al. 2007

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The function of the stacking tryptophan, W290, a second coordination sphere residue in galactose oxidase has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and x -ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly lower in the W290 variants. The K m for D-galactose for W290H is similar to wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding/positioning via the -NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radical in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has similar properties to the wild type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semi-reduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes the variants are also closely similar to the wild type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.Over the past twenty years, there has been a growing appreciation for the catalytic utility of protein-derived free radical cofactors in enzymes (1-3). Free radical chemistry is harnessed to catalyze bond activation and molecular rearrangements in a wide variety of enzymes including ribonucleotide reductase (4-7), DNA photolyase (8), cytochrome c peroxidase (9), pyruvateformate lyase (10), lysine-2,3-aminomutase (11), prostaglandin H synthase (12), glyoxal oxidase (13), and galactose oxidase (14).*Authors to whom correspondence should be addressed. Email: dmdooley@montana.edu, Tel: 406-994-4373, FAX: 406 -994-7989; Email: m.j.mcpherson@leeds.ac.uk, Tel: +44 113 233-2595, FAX: +44 113 233-3167. 1 Data deposition: The atomic coordinates and structure factors for W290G, W290F and W290H have been deposited in the Protein Data Bank, www.rcsb.org. † This work was supported by a grant from the National Institutes of Health (GM27659 DMD) and from the Biotechnology and Biological Sciences Research Council (MJM). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIt...

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“…Copper amine oxidases (CAOs) catalyse the oxidative deamination of primary amines into their corresponding aldehydes and exert fundamental functions in a wealth of aerobic organisms from bacteria to yeast, plants, and mammals. 5–7 CAOs in microorganisms have a nutritional role in catabolizing primary amines. 7 In higher eukaryotes, CAOs in animals participate in the degradation of bio-active amines, and regulation of cell adhesion, cell death, and collagen cross-linking.…”

Section: Introductionmentioning

confidence: 99%

Molecular mechanism of a large conformational change of the quinone cofactor in the semiquinone intermediate of bacterial copper amine oxidase

et al. 2022

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Galactose Oxidase

Cited by 4 publications

References 56 publications

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

The Stacking Tryptophan of Galactose Oxidase: A Second-Coordination Sphere Residue that Has Profound Effects on Tyrosyl Radical Behavior and Enzyme Catalysis^,

Molecular mechanism of a large conformational change of the quinone cofactor in the semiquinone intermediate of bacterial copper amine oxidase

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Galactose Oxidase

Cited by 4 publications

References 56 publications

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

Cross-Link Formation of the Cysteine 228−Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does Not Require Dioxygen

The Stacking Tryptophan of Galactose Oxidase: A Second-Coordination Sphere Residue that Has Profound Effects on Tyrosyl Radical Behavior and Enzyme Catalysis,

Molecular mechanism of a large conformational change of the quinone cofactor in the semiquinone intermediate of bacterial copper amine oxidase

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The Stacking Tryptophan of Galactose Oxidase: A Second-Coordination Sphere Residue that Has Profound Effects on Tyrosyl Radical Behavior and Enzyme Catalysis^,