The molecular properties of the copper enzyme galactose oxidase

Kosman, Daniel J.; Ettinger, Murray J.; Weiner, Ronald E.; Massaro, Edward J.

doi:10.1016/0003-9861(74)90271-9

Cited by 92 publications

(73 citation statements)

References 35 publications

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“…Galactose oxidase purified from the yeast expression system had essentially indistinguishable kinetic and structural properties from the enzyme isolated from the fungal expression system. Protein concentrations were calculated using ε 280 of 104900 M −1 cm −1 (50). The concentrations of W290 variants were corrected by multiplying by 16/15 to account for the lowered tryptophan content.…”

Section: Experimental Procedures (Materials and Methods) Enzyme Purifmentioning

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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Section: Experimental Procedures (Materials and Methods) Enzyme Purifmentioning

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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“…[1][2][3][4][5][6][7][8][9][10][11] This two electron chemistry is promoted by a single copper atom, working in synergy with a tyrosyl radical from the protein. Galactose Oxidase possesses a N 2 O 2 donor set: One oxygen atom, from the Tyr 272 · radical residue, is the organic redox center, while the other (from Tyr 495 ) controls the proton transfer process.…”

Section: Introductionmentioning

confidence: 99%

Galactose Oxidase Models: Creation and Modification of Proton Transfer Coupled to Copper(II) Coordination Processes in Pro‐Phenoxyl Ligands

et al. 2006

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Two tripodal ligands containing two pyridyl and one 4‐(benzimidazol‐2‐yl)‐2‐tert‐butylphenol (HLH) and one 2‐tert‐butyl‐4‐(N‐methylbenzimidazol‐2‐yl)phenol (HLMe) unit have been synthesized. They possess a N3O donor set that is known to stabilize phenoxyl radicals more efficiently than the corresponding N2O2 donor unit. Reaction of one molar equiv. of Cu(ClO4)2·6H2O with HLH or HLMe affords the zwitterionic benzimidazolium phenolate complexes [CuII(HLH)]2+ and [CuII(HLMe)]2+ via a proton transfer reaction coupled to copper(II) coordination. Addition of HClO4 to [CuII(HLH)]2+ and [CuII(HLMe)]2+ results in the formation of the benzimidazolium phenol complexes [CuII(H2LH)]3+ and [CuII(H2LMe)]2+, while addition of NEt3 affords the benzimidazol‐phenolate complexes [CuII(LH)]2+ and [CuII(LMe)]2+, respectively. The phenol’s pKa is remarkably low due to the strong withdrawing effect of the benzimidazolium substituent. X‐ray crystallographic analysis of the copper(II) complexes shows that deprotonation of the axial phenol forces the metal to move out of the square plane towards the oxygen atom, and one (or two) Cu–Npyridine equatorial bond length increases. The copper(II) phenoxyl species [CuII(HLH)]·3+ and [CuII(HLMe)]·3+ were prepared electrochemically, or by addition of two molar equiv. of copper(II) to HLH or HLMe. Under these conditions, radical formation has never been observed for tripodal ligands containing two pyridyl and one 2,4‐di‐tert‐butylphenol group. This difference is explained in terms of the proton transfer mechanism and redox potentials. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

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“…is a copper-containing enzyme that catalyses the oxidation of primary alcohols to their corresponding aldehydes, a two-electron reaction, but with only a single copper at the active site (1). The second redox active center necessary for the reaction was found to be situated at a tyrosine residue (2).…”

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confidence: 99%

Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

Firbank

Rogers

Wilmot

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

112

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Galactose oxidase (EC 1.1.3.9) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa prosequence at the N terminus. Previous work has shown that the aerobic addition of Cu 2؉ to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 Å, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.

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The molecular properties of the copper enzyme galactose oxidase

Cited by 92 publications

References 35 publications

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

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

Galactose Oxidase Models: Creation and Modification of Proton Transfer Coupled to Copper(II) Coordination Processes in Pro‐Phenoxyl Ligands

Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

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The molecular properties of the copper enzyme galactose oxidase

Cited by 92 publications

References 35 publications

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

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

Galactose Oxidase Models: Creation and Modification of Proton Transfer Coupled to Copper(II) Coordination Processes in Pro‐Phenoxyl Ligands

Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

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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^,

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