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Structure and mechanism of galactose oxidase. The free radical site
Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, a...
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| Published in: | The Journal of biological chemistry 1994-10, Vol.269 (40), p.25095-25105 |
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| Main Authors: | , , , , , , , , |
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| cited_by | cdi_FETCH-LOGICAL-c496t-945c8f048c90bac0663f98e13b937610665b240126be857e053e67a6e05268713 |
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| cites | cdi_FETCH-LOGICAL-c496t-945c8f048c90bac0663f98e13b937610665b240126be857e053e67a6e05268713 |
| container_end_page | 25105 |
| container_issue | 40 |
| container_start_page | 25095 |
| container_title | The Journal of biological chemistry |
| container_volume | 269 |
| creator | Baron, A.J. (The University of Leeds, Leeds, UK.) Stevens, C Wilmot, C Seneviratne, K.D Blakeley, V Dooley, D.M Phillips, S.E.V Knowles, P.F McPherson, M.J |
| description | Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site |
| doi_str_mv | 10.1016/s0021-9258(17)31504-1 |
| format | article |
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The free radical site</title><source>ScienceDirect®</source><creator>Baron, A.J. (The University of Leeds, Leeds, UK.) ; Stevens, C ; Wilmot, C ; Seneviratne, K.D ; Blakeley, V ; Dooley, D.M ; Phillips, S.E.V ; Knowles, P.F ; McPherson, M.J</creator><creatorcontrib>Baron, A.J. (The University of Leeds, Leeds, UK.) ; Stevens, C ; Wilmot, C ; Seneviratne, K.D ; Blakeley, V ; Dooley, D.M ; Phillips, S.E.V ; Knowles, P.F ; McPherson, M.J</creatorcontrib><description>Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. 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(The University of Leeds, Leeds, UK.)</creatorcontrib><creatorcontrib>Stevens, C</creatorcontrib><creatorcontrib>Wilmot, C</creatorcontrib><creatorcontrib>Seneviratne, K.D</creatorcontrib><creatorcontrib>Blakeley, V</creatorcontrib><creatorcontrib>Dooley, D.M</creatorcontrib><creatorcontrib>Phillips, S.E.V</creatorcontrib><creatorcontrib>Knowles, P.F</creatorcontrib><creatorcontrib>McPherson, M.J</creatorcontrib><title>Structure and mechanism of galactose oxidase. The free radical site</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site</description><subject>ACIDE AMINE</subject><subject>ACTIVIDAD ENZIMATICA</subject><subject>ACTIVITE ENZYMATIQUE</subject><subject>AMINOACIDOS</subject><subject>ASPERGILLUS NIDULANS</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>COBRE</subject><subject>Crystallography, X-Ray</subject><subject>CUIVRE</subject><subject>Free Radicals</subject><subject>Fusarium</subject><subject>GALACTOSA</subject><subject>GALACTOSE</subject><subject>Galactose Oxidase - biosynthesis</subject><subject>Galactose Oxidase - chemistry</subject><subject>Galactose Oxidase - isolation & purification</subject><subject>HYPOMYCES</subject><subject>Kinetics</subject><subject>MINERALOGIA</subject><subject>MINERALOGIE</subject><subject>Molecular Sequence Data</subject><subject>MUTACION</subject><subject>MUTATION</subject><subject>OXIDORREDUCTASAS</subject><subject>OXYDOREDUCTASE</subject><subject>Polymerase Chain Reaction</subject><subject>RADICAL LIBRE</subject><subject>RADICALES LIBRES</subject><subject>TECHNIQUE ANALYTIQUE</subject><subject>TECNICAS ANALITICAS</subject><subject>TRANSFORMACION GENETICA</subject><subject>TRANSFORMATION GENETIQUE</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><recordid>eNqFkEtr3DAQgEVoSLeb_IFAQIdS2oMTjZ7WMSx9BBZy2CzkJmTteK1gr1PJpum_r_dBrtVlNMw3Dz5CboDdAgN9lxnjUFiuyq9gvglQTBZwRmbASlEIBc8fyOwd-Ug-5fzCpictXJALY7kFW87IYjWkMQxjQup3G9phaPwu5o72Nd361oehz0j7t7jxGW_pU4O0Tog0-U0MvqU5DnhJzmvfZrw6xTlZ__j-tPhVLB9_Pizul0WQVg-FlSqUNZNlsKzygWktalsiiMoKo2HKVcUlA64rLJVBpgRq4_X04bo0IObky3Hua-p_j5gH18UcsG39DvsxO6ON4orx_4IwrZbqMFEdwZD6nBPW7jXFzqe_DpjbW3arvUK3V-jAuINlt--7OS0Yqw43710nrVP987HexG3zJyZ0VexDg53j2jrJ3HSmVRN2fcRq3zu_TTG79coqpSVw8Q8cpYmE</recordid><startdate>19941007</startdate><enddate>19941007</enddate><creator>Baron, A.J. 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(The University of Leeds, Leeds, UK.) ; Stevens, C ; Wilmot, C ; Seneviratne, K.D ; Blakeley, V ; Dooley, D.M ; Phillips, S.E.V ; Knowles, P.F ; McPherson, M.J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c496t-945c8f048c90bac0663f98e13b937610665b240126be857e053e67a6e05268713</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>ACIDE AMINE</topic><topic>ACTIVIDAD ENZIMATICA</topic><topic>ACTIVITE ENZYMATIQUE</topic><topic>AMINOACIDOS</topic><topic>ASPERGILLUS NIDULANS</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>COBRE</topic><topic>Crystallography, X-Ray</topic><topic>CUIVRE</topic><topic>Free Radicals</topic><topic>Fusarium</topic><topic>GALACTOSA</topic><topic>GALACTOSE</topic><topic>Galactose Oxidase - biosynthesis</topic><topic>Galactose Oxidase - chemistry</topic><topic>Galactose Oxidase - isolation & purification</topic><topic>HYPOMYCES</topic><topic>Kinetics</topic><topic>MINERALOGIA</topic><topic>MINERALOGIE</topic><topic>Molecular Sequence Data</topic><topic>MUTACION</topic><topic>MUTATION</topic><topic>OXIDORREDUCTASAS</topic><topic>OXYDOREDUCTASE</topic><topic>Polymerase Chain Reaction</topic><topic>RADICAL LIBRE</topic><topic>RADICALES LIBRES</topic><topic>TECHNIQUE ANALYTIQUE</topic><topic>TECNICAS ANALITICAS</topic><topic>TRANSFORMACION GENETICA</topic><topic>TRANSFORMATION GENETIQUE</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Baron, A.J. 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(The University of Leeds, Leeds, UK.)</au><au>Stevens, C</au><au>Wilmot, C</au><au>Seneviratne, K.D</au><au>Blakeley, V</au><au>Dooley, D.M</au><au>Phillips, S.E.V</au><au>Knowles, P.F</au><au>McPherson, M.J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structure and mechanism of galactose oxidase. The free radical site</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1994-10-07</date><risdate>1994</risdate><volume>269</volume><issue>40</issue><spage>25095</spage><epage>25105</epage><pages>25095-25105</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. 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| ispartof | The Journal of biological chemistry, 1994-10, Vol.269 (40), p.25095-25105 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | ScienceDirect® |
| subjects | ACIDE AMINE ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE AMINOACIDOS ASPERGILLUS NIDULANS Base Sequence Binding Sites COBRE Crystallography, X-Ray CUIVRE Free Radicals Fusarium GALACTOSA GALACTOSE Galactose Oxidase - biosynthesis Galactose Oxidase - chemistry Galactose Oxidase - isolation & purification HYPOMYCES Kinetics MINERALOGIA MINERALOGIE Molecular Sequence Data MUTACION MUTATION OXIDORREDUCTASAS OXYDOREDUCTASE Polymerase Chain Reaction RADICAL LIBRE RADICALES LIBRES TECHNIQUE ANALYTIQUE TECNICAS ANALITICAS TRANSFORMACION GENETICA TRANSFORMATION GENETIQUE |
| title | Structure and mechanism of galactose oxidase. The free radical site |
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