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Structural Studies of Catalysis and Processing in Galactose

By Nana Akumanyi


The fungal copper containing enzyme galactose oxidase is one of the earliest examples of a protein derived radical cofactor. A covalent bond, formed between the active site cysteine and the Cc of a tyrosine act as a site for radical formation, allowing the enzyme to catalyse the two electron oxidation of primary alcohols to their corresponding aldehydes, with the coproduction of hydrogen peroxide.\ud \ud The formation of the post-translationally modified redox cofactor is an autocatalytic process in vitro, requiring only the aerobic addition of copper. A premature form of galactose oxidase, lacking the cofactor has been crystallised and the structure determined. Copper incubation of this protein has been carried out and the structures determined in order to characterise processing intermediates and the mechanism of cofactor biogenesis.\ud \ud The mature, fully processed and catalytically active enzyme contains a radical which is thought to be stabilised by the active site environment, particularly a tryptophan residue\ud which stacks over the cofactor site. The structure of a galactose oxidase variant in which the tryptophan is mutated to a glycine has been determined and characterised.\ud \ud Past attempts to obtain an enzyme-substrate complex with sugar molecules have been unsuccessful since space at the active site of the original crystal form was limited.\ud Additionally crystals have not been activated prior to substrate incubation. Investigation of substrate binding using activated crystals and a different crystal form have been carried out. The C383S variant protein, has been shown to have improved activity against galactose, however, the wide substrate specificity of the enzyme has allowed investigation of substrate binding using a smaller substrate. Two enzyme-substrate structures, determined to 1.8 A and 1.9 A resolution have be obtained following incubation of C383S with a smaller alcohol substrate.\ud \ud Investigation of the fully reduced form of galactose oxidase has proved difficult due to the\ud high reactivity of Cu'+ with oxygen. Apo-galactose oxidase crystals were anaerobically incubated with Cu" and the structures determined in order to characterise the fully reduced form of galactose oxidase

Publisher: Institute for Molecular and Cellular Biology (Leeds)
Year: 2007
OAI identifier: oai:etheses.whiterose.ac.uk:649

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  11. (2002). Dissecting an enzyme: Model compounds for the galactose oxidase radical site, doi
  12. (1992). Electron paramagnetic resonance and electron nuclear double resonance spectroscopies of the radical site in galactose oxidase and of thioether-substituted phenol model compounds, doi
  13. (2004). Electrostatic control of the tryptophan radical in cytochrome c peroxidase. doi
  14. (2004). Enhanced fructose oxidase activity in a galactose oxidase variant, doi
  15. (2001). Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution, Protein Eng. doi
  16. (1963). Galactose oxidase of Polyporus circinatus: A copper enzyme,
  17. (2001). Galactose Oxidase, doi
  18. (1996). Glyoxal oxidase from Phanerochaete chrysosporium is a new radical-copper oxidase, doi
  19. (1996). High-frequency electron Rogers et al. paramagnetic resonance spectroscopy of the apogalactose oxidase radical, doi
  20. (1989). Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES, doi
  21. (1973). Indirect coulometric titration of biological electron transport components, doi
  22. (2002). Influence of the protein en ironment on the properties of a tyrosyl radical in reaction centers from Rhodobacter sphaeroides, doi
  23. (2001). Interconversion of Cu' and Cu" forms of galactose oxidase: Comparison of reduction potentials, doi
  24. (1998). Kinetic isotope effects as probes of the mechanism of galactose oxidase, doi
  25. (1993). Ligand interactions with galactose oxidase: Mechanistic insights, Binphv c. doi
  26. (1996). lolecular modeling studies on oxidation of hexopyranoses by galactose oxidase. An active site topology apparently designed to catalyze radical reactions, either concerted or stepwise, doi
  27. (2002). Modification of galactose oxidase to introduce glucose 6-oxidase activity, doi
  28. (2000). Novel cofactors via post-translational modifications of enzyme active sites, doi
  29. (1991). Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase, doi
  30. (2001). Posttranslationally modified tyrosines from galactose oxidase and cytochrome c oxidase, Adv. Protein Chem. doi
  31. (1997). Processing of X-ray diffraction data collected in oscillation mode, Methods En - doi
  32. (1997). Properties of the Trp290His variant ofFusarium NRRL 2903 galactose oxidase: Interactions of the GOase,,, state with different buffers, its redox activity and ability to bind azide, doi
  33. (1989). Protein radical involvement in biological catalysis, doi
  34. (1998). Protein radicals in enzyme catalysis, doi
  35. (2003). Radical initiation in the class I ribonucleotide reductase: Longrange proton-coupled electron transfer'' doi
  36. (2001). Radical mechanisms of enzymatic catalysis,
  37. (1997). Reactivity of the tyrosyl radical of Escherichia coli ribonucleotide reductase: Control by the protein. doi
  38. (1985). Redox activation of galactose oxidase: Thin-layer electrochemical study. doi
  39. (1995). Residues important for radical stability in ribonucleotide reductase from Escherichia coli,
  40. (1989). Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase,
  41. (1995). Resonance raman spectroscopy of galactose oxidase: A new interpretation based on model compound free radical spectra, doi
  42. (1983). Ribonucleotide reductase: A radical enzyme, doi
  43. (2004). Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase, doi
  44. (2002). Second-sphere contributions to substrate-analogue binding in iron(III) superoxide dismutase, doi
  45. (2000). Spectroscopic and magnetochemieal studies on the active site copper complex in galactose oxidase. doi
  46. (1995). Spectroscopic studies of galactose oxidase, in Redox-active Amino Acids in doi
  47. (1995). Spectroscopic studies of the active site of galactose oxidase. doi
  48. (1998). SPOCK: The structural properties observation and calculation kit,
  49. (2001). Stable glycyl radical from pyruvate formate-lyase and ribonucleotide reductase(III). -Id" Protein Chem. doi
  50. (2001). Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase1, doi
  51. (1974). Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins, doi
  52. (2002). Structural investigation of processing and catalysis in galactose oxidase,
  53. (2001). Structure and function cif the radical enzyme ribonucleotide reductase, doi
  54. (2003). Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors, doi
  55. (1997). Structure and mechanism of galactose oxidasc Catalytic role of tyrosine 495, doi
  56. (1994). Structure and mechanism of galactose oxidase. The free radical site,
  57. (2006). Structure of the oxidized active site of galactose oxidase from realistic in silico models, doi
  58. (1996). Synthesis, structure and properties of a model for galactose oxidase, doi
  59. (1998). tbfB, a gene encoding a putative galactose oxidase, is involved in Stigmatella aurantiaca fruiting body formation,
  60. (1988). The active site of galactose oxidase. doi
  61. (2005). The active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylase, doi
  62. (1962). The D-galactose oxidase of Polyporus circinatus,
  63. (1994). The free radical-coupled copper active site of galactose oxidase, in Metalloenrymes involving amino acid residues and related
  64. (1971). The interpretation of protein structures: Estimation of static accessibility, doi
  65. (1974). The molecular properties of the copper enzyme galactose oxidase, doi
  66. (1936). The oxidation potential of the system potassium molybdocyanide-molybdocyanide and the effect of neutral salts on the potential, doi
  67. (1998). The protein environment surrounding tyrosyl radicals D' and Z' in photosystem 11: A difference Fourier-transform infrared spectroscopic study, doi
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  69. (1999). The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by r-. 7r interactions, doi
  70. (1992). Thrctidimensional structure of galactose oxidase: An enzyme with a built-in secondary cofactor, Faraday Discuss. doi
  71. (2000). Tyrosyl radicals doi
  72. (2005). Versatile peroxidase oxidation of high redox potential aromatic compounds: Site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways. doi
  73. (1999). X-ray crystallographic studies on coppercontaining oxidases,

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