MIT Libraries homeMIT Libraries logoDSpace@MIT

MIT
View Item 
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

Detection of Covalent and Noncovalent Intermediates in the Polymerization Reaction Catalyzed by a C149S Class III Polyhydroxybutyrate Synthase

Author(s)
Li, Ping; Chakraborty, Sumit; Stubbe, JoAnne
Thumbnail
DownloadStubbe_Detection of covalent.pdf (1.896Mb)
PUBLISHER_POLICY

Publisher Policy

Article 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.

Terms of use
Article 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.
Metadata
Show full item record
Abstract
Polyhydroxybutyrate (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.
Date issued
2009-08
URI
http://hdl.handle.net/1721.1/82557
Department
Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Li, 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.
Version: Author's final manuscript
ISSN
0006-2960
1520-4995

Collections
  • MIT Open Access Articles

Browse

All of DSpaceCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

My Account

Login

Statistics

OA StatisticsStatistics by CountryStatistics by Department
MIT Libraries homeMIT Libraries logo

Find us on

Twitter Instagram YouTube

MIT Libraries navigation

SearchHours & locationsBorrow & requestResearch supportAbout us
PrivacyPermissionsAccessibility
MIT
Massachusetts Institute of Technology
Content created by the MIT Libraries, CC BY-NC unless otherwise noted. Notify us about copyright concerns.