| Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase. | |
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MedLine Citation:
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PMID: 19916558 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Escherichia coli ribonucleotide reductase (RNR), an alpha2beta2 complex, catalyzes the conversion of nucleoside 5'-diphosphate substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). beta2 contains the essential diferric tyrosyl radical (Y(122)(*)) cofactor which, in the presence of S and E, oxidizes C(439) in alpha to a thiyl radical, C(439)(*), to initiate nucleotide reduction. This oxidation occurs over 35 A and is proposed to involve a specific pathway: Y(122)(*) --> W(48) --> Y(356) in beta2 to Y(731) --> Y(730) --> C(439) in alpha2. 3-Aminotyrosine (NH(2)Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH(2)Y(*)) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y(356)F-beta2:Y(731)NH(2)Y-alpha2, Y(356)F-beta2:Y(730)NH(2)Y-alpha2, and wt-beta2:Y(731)F/Y(730)NH(2)Y-alpha2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH(2)Y(*) was detected. To study off-pathway oxidation, Y(413), located 5 A from Y(730) and Y(731) but not implicated in long-range oxidation, was examined. Evidence for NH(2)Y(413)(*) was sought in three complexes: wt-beta2:Y(413)NH(2)Y-alpha2 (a), wt-beta2:Y(731)F/Y(413)NH(2)Y-alpha2 (b), and Y(356)F-beta2:Y(413)NH(2)Y-alpha2 (c). With (a), NH(2)Y(*) was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH(2)Y(*) formation was 0.2-0.3% of that observed at 731 and 730, and with (c), no NH(2)Y(*) was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C(439). |
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Authors:
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Ellen C Minnihan; Mohammad R Seyedsayamdost; JoAnne Stubbe |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biochemistry Volume: 48 ISSN: 1520-4995 ISO Abbreviation: Biochemistry Publication Date: 2009 Dec |
Date Detail:
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Created Date: 2009-12-22 Completed Date: 2010-01-12 Revised Date: 2011-08-01 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 12125-32 Citation Subset: IM |
Affiliation:
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Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,Massachusetts 02139-4307, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Electron Spin Resonance Spectroscopy Electron Transport Escherichia coli / chemistry*, genetics, metabolism Escherichia coli Proteins / chemistry*, genetics, metabolism Free Radicals / chemistry* Oxidation-Reduction Ribonucleoside Diphosphate Reductase / chemistry*, genetics, metabolism Tyrosine / analogs & derivatives*, chemistry |
| Grant Support | |
ID/Acronym/Agency:
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GM29595/GM/NIGMS NIH HHS; R01 GM029595-30/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Escherichia coli Proteins; 0/Free Radicals; 300-34-5/3-aminotyrosine; 55520-40-6/Tyrosine; EC 1.17.4.1/Ribonucleoside Diphosphate Reductase |
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