Dissertation
Biochemical studies on plant ADP-glucose pyrophosphorylase regulatory properties
Washington State University
Doctor of Philosophy (PhD), Washington State University
12/2007
DOI:
https://doi.org/10.7273/000005691
Abstract
ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of
starch synthesis in plants. Plant AGPases are composed of two distinct subunits, a
catalytic small subunit (SS) and a non-catalytic large subunit (LS). Previously, we have
identified and characterized an allosteric LSP52L mutant, which when co-expressed with
wild type SS, formed an enzyme with down-regulating allosteric properties. To further
investigate the structure-function relationships between the two subunits with regard to
allosteric regulation, random chemical mutagenesis was performed to generate SS
suppressors of LSP52L. Several putative SS mutant suppressors were identified by their
ability to restore glycogen accumulation when co-expressed with LSP52L mutant in
Escherichia coli glgC- strain. Kinetic analysis of these second-site mutant SS enzymes
indicated that they comprise two distinct classes based on the SS interaction with LSP52L
or wild type LS. One class contained bona fide SS suppressors (SSL46F and SSP112L),
which reversed the down-regulatory properties of LSP52L but not to wild type LS. The
other SS class contained allosteric mutants, SSP308L and SSR350K, which generated upregulated
enzymes with wild type LS as well as LSP52L. These results indicate that both LS and SS have a regulatory function in controlling allosteric properties through enhancing subunit interactions. In addition to allosteric regulation, plant AGPases are redox regulated by reduction of an intermolecular disulfide bond (S-S) between two Cys12 residues of catalytic small subunits (SSs). In this study, we replaced the Cys 12 residues with Ala (SSC12A) to remove the disulfide bond in both the potato tuber and Arabidopsis leaf AGPase SSs. SSC12As were co-expressed with the corresponding LSs and resulting enzymes were purified. Kinetic analysis of SSC12A containing enzymes indicated that they have high affinity to the activator 3-phosphoglycerate and substrate ATP in both reducing and oxidizing conditions. Thermal stability of these enzymes was also investigated using both kinetics and biochemical approaches. SSC12A containing
enzymes were less stable than the wild type enzymes at high temperatures, based on the
results from circular dichroism spectroscopy and enzyme activity. Our results suggested
the S-S bond in the AGPase is important for not only enhancing enzyme activation
through higher affinity to activator and substrate, but also enzyme stability to maintain
ordered structure at high temperatures.
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Details
- Title
- Biochemical studies on plant ADP-glucose pyrophosphorylase regulatory properties
- Creators
- Dongwook Kim
- Contributors
- Thomas W Okita (Chair) - Washington State University, Institute of Biological ChemistryGerald E Edwards (Committee Member) - Washington State University, School of Biological SciencesNancy S Magnuson (Committee Member) - Washington State University, School of Molecular Biosciences
- Awarding Institution
- Washington State University
- Academic Unit
- School of Molecular Biosciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 75
- Identifiers
- 99901054938201842
- Language
- English
- Resource Type
- Dissertation