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dc.contributor.authorBuckley, Rachael Marie
dc.contributor.authorStubbe, JoAnne
dc.date.accessioned2016-08-18T20:40:38Z
dc.date.available2016-08-18T20:40:38Z
dc.date.issued2015-03
dc.date.submitted2015-02
dc.identifier.issn0006-2960
dc.identifier.issn1520-4995
dc.identifier.urihttp://hdl.handle.net/1721.1/103958
dc.description.abstractPolyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaC[subscript Cc]) is a highly soluble protein with a turnover number of 75 s[superscript –1] and no lag phase in coenzyme A (CoA) release. Studies with [1-[superscript 14]C]HBCoA and PhaC[subscript Cc] monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [[superscript 3]H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaC[subscript Cc] with [1-[superscript 14]C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s[superscript –1]. We propose that the [[superscript 3]H]sDCoA forms via attack of CoA on the oxoester of the [[superscript 3]H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC[subscript Cc], and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation.en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (NIH Grant GM49171)en_US
dc.language.isoen_US
dc.publisherAmerican Chemical Society (ACS)en_US
dc.relation.isversionofhttp://dx.doi.org/10.1021/bi501405ben_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.sourceACSen_US
dc.titleChemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Terminationen_US
dc.typeArticleen_US
dc.identifier.citationRachael M. Buckley, and JoAnne Stubbe. "Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination." Biochemistry 54:12 (2015), pp. 2117-2125. © 2015 American Chemical Society.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.mitauthorBuckley, Rachael Marieen_US
dc.contributor.mitauthorStubbe, JoAnneen_US
dc.relation.journalBiochemistryen_US
dc.eprint.versionFinal published versionen_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.embargo.termsNen_US
dc.identifier.orcidhttps://orcid.org/0000-0001-8076-4489
mit.licensePUBLISHER_POLICYen_US


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