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dc.contributor.authorLi, Ping
dc.contributor.authorChakraborty, Sumit
dc.contributor.authorStubbe, JoAnne
dc.date.accessioned2013-11-22T19:35:06Z
dc.date.available2013-11-22T19:35:06Z
dc.date.issued2009-08
dc.date.submitted2009-08
dc.identifier.issn0006-2960
dc.identifier.issn1520-4995
dc.identifier.urihttp://hdl.handle.net/1721.1/82557
dc.description.abstractPolyhydroxybutyrate (PHB) synthases catalyze the conversion of 3-hydroxybutyryl coenzyme A (HBCoA) to PHB with a molecular mass of 1.5 MDa. The class III synthase from Allochromatium vinosum is a tetramer of PhaEPhaC (each 40 kDa). The polymerization involves covalent catalysis using C149 of PhaC with one PHB chain per PhaEC dimer. Two mechanisms for elongation have been proposed. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer. The second involves C149 and covalent and noncovalent (HB)[subscript n]CoA intermediates. Two approaches were investigated to distinguish between these models. The first involved the wild-type (wt) PhaEC primed with sTCoA [a CoA ester of (HB)[subscript 3] in which the terminal HO group is replaced with an H] which uniformly loads the enzyme. The primed synthase was reacted with [1-[superscript 14]C]HBCoA by a rapid chemical quench method and analyzed for covalent and noncovalent intermediates. Radiolabel was found only with the protein. The second approach used C149S-PhaEC which catalyzes polymer formation at 1/2200 of the rate of wt-PhaEC (1.79 min[superscript −1] vs 3900 min[superscript −1]). C149S-PhaEC was incubated with [1-[superscript 14]C]HBCoA and chemically quenched on the minute time scale to reveal noncovalently bound [1-[superscript 14]C](HB)[subscript 2]CoA and (HB)[subscript 3]CoA as well as covalently labeled protein. Synthesized (HB)[subscript n]CoA (n = 2 or 3) was shown to acylate PhaEC with rate constants of 1−2 min[superscript −1], and these species were converted into polymer. Thus, the (HB)[subscript n]CoA analogues function as kinetically and chemically competent intermediates. These results support the mechanism involving covalently and noncovalently bound intermediates.en_US
dc.description.sponsorshipNational Institutes of Health (U.S.) (Grant GM49171)en_US
dc.description.sponsorshipNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (Postdoctoral Fellowship F32GM082067)en_US
dc.language.isoen_US
dc.publisherAmerican Chemical Society (ACS)en_US
dc.relation.isversionofhttp://dx.doi.org/10.1021/bi901329ben_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.titleDetection of Covalent and Noncovalent Intermediates in the Polymerization Reaction Catalyzed by a C149S Class III Polyhydroxybutyrate Synthaseen_US
dc.typeArticleen_US
dc.identifier.citationLi, Ping, Sumit Chakraborty, and JoAnne Stubbe. “Detection of Covalent and Noncovalent Intermediates in the Polymerization Reaction Catalyzed by a C149S Class III Polyhydroxybutyrate Synthase.” Biochemistry 48, no. 39 (October 6, 2009): 9202-9211.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.mitauthorLi, Pingen_US
dc.contributor.mitauthorChakraborty, Sumiten_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.orderedauthorsLi, Ping; Chakraborty, Sumit; Stubbe, JoAnneen_US
dc.identifier.orcidhttps://orcid.org/0000-0001-8076-4489
mit.licensePUBLISHER_POLICYen_US
mit.metadata.statusComplete


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