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Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.ORCID iD: 0000-0001-8685-9665
2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 1, 295-300 p.Article in journal (Refereed) Published
Abstract [en]

Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117-138). The C99S mutant had about half the V (max) of the wild-type and a 12-fold higher K (m) for PP(i), whereas NIN and LL mutations lowered the V (max) by 12- and 2-fold, respectively, with relatively small effects on substrate K (m) values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.

Place, publisher, year, edition, pages
Portland Press , 2002. Vol. 367, no 1, 295-300 p.
Keyword [en]
cytosol, site-directed mutant, sucrose metabolism, sucrose synthesis
Identifiers
URN: urn:nbn:se:umu:diva-41816DOI: 10.1042/BJ20020772PubMedID: 12088504OAI: oai:DiVA.org:umu-41816DiVA: diva2:407860
Available from: 2011-04-01 Created: 2011-04-01 Last updated: 2017-12-11Bibliographically approved

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Martz, FrançoiseWilczynska, MalgorzataKleczkowski, Leszek A
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