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dc.contributor.authorArtin, Erin
dc.contributor.authorWang, Jun
dc.contributor.authorLohman, Gregory J. S.
dc.contributor.authorYokoyama, Kenichi
dc.contributor.authorYu, Guixue
dc.contributor.authorBar, Galit
dc.contributor.authorStubbe, JoAnne
dc.contributor.authorGriffin, Robert Guy
dc.date.accessioned2013-11-08T20:27:34Z
dc.date.available2013-11-08T20:27:34Z
dc.date.issued2009-11
dc.date.submitted2009-11
dc.identifier.issn0006-2960
dc.identifier.issn1520-4995
dc.identifier.urihttp://hdl.handle.net/1721.1/82066
dc.description.abstractGemcitabine 5′-diphosphate (F[subscript 2]CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an α2β2 complex, when incubated with 1 equiv of F[subscript 2]CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of α with the sugar of F[subscript 2]CDP (one label/α2β2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that α migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-α in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-α migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR’s active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1′-[superscript 2]H]-F[subscript 2]CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of g[subscript x] 2.00738, g[subscript y] 2.00592, and g[subscript z] 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1′-[superscript 1]H]-F[subscript 2]CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (Grant GM29595)en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (Grant EB-002804)en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (Grant EB-002026)en_US
dc.language.isoen_US
dc.publisherAmerican Chemical Society (ACS)en_US
dc.relation.isversionofhttp://dx.doi.org/10.1021/bi901590qen_US
dc.rightsArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.en_US
dc.sourcePMCen_US
dc.titleInsight into the Mechanism of Inactivation of Ribonucleotide Reductase by Gemcitabine 5′-Diphosphate in the Presence or Absence of Reductanten_US
dc.typeArticleen_US
dc.identifier.citationArtin, Erin, Jun Wang, Gregory J. S. Lohman, Kenichi Yokoyama, Guixue Yu, Robert G. Griffin, Galit Bar, and JoAnne Stubbe. “Insight into the Mechanism of Inactivation of Ribonucleotide Reductase by Gemcitabine 5′-Diphosphate in the Presence or Absence of Reductant.” Biochemistry 48, no. 49 (December 15, 2009): 11622-11629.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.mitauthorArtin, Erinen_US
dc.contributor.mitauthorWang, Junen_US
dc.contributor.mitauthorLohman, Gregory J. S.en_US
dc.contributor.mitauthorYokoyama, Kenichien_US
dc.contributor.mitauthorYu, Guixueen_US
dc.contributor.mitauthorGriffin, Robert Guyen_US
dc.contributor.mitauthorBar, Galiten_US
dc.contributor.mitauthorStubbe, JoAnneen_US
dc.relation.journalBiochemistryen_US
dc.eprint.versionAuthor's final manuscripten_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsArtin, Erin; Wang, Jun; Lohman, Gregory J. S.; Yokoyama, Kenichi; Yu, Guixue; Griffin, Robert G.; Bar, Galit; Stubbe, JoAnneen_US
dc.identifier.orcidhttps://orcid.org/0000-0003-1589-832X
dc.identifier.orcidhttps://orcid.org/0000-0001-8076-4489
mit.licensePUBLISHER_POLICYen_US


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