Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of
glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to
the non-reducing ends of glycogen and its activity is negatively regulated by
phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A
highly conserved cluster of six arginine residues on the C-terminal domain
controls the responses toward these opposing signals. Previous studies had
shown that tetrameric enzyme exists in three conformational states which are
linked to specific structural changes in the regulatory helices that carry the cluster
of arginines. These helices are found opposite and anti-parallel to one another at
one of the subunit interfaces. The binding of G6P beneath the regulatory helices
induces large scale conformational changes which open up the catalytic cleft for
better substrate access. We solved the crystal structure of the enzyme in its
inhibited state and found that the tetrameric and regulatory interfaces are more
compacted compared to other states. The structural consequence of the tighter
interfaces within the inhibited state of the tetramer is to lower the ability of
glycogen chains to access to the catalytic cleft. Based on these observations, we
developed a novel regulatory feature in yeast GS by substituting two of its
conserved arginine residues on the regulatory helix with cysteines that permits its
activity to be controlled by reversible oxidation/reduction of the cysteine residues
which mimics the effects of reversible phosphorylation. In addition to defining the
structural changes that give rise to the inhibited states, we also used X-ray
crystallography to define the mechanism by which the enzyme discriminates
between different UDP-sugar donors to be used as substrates in the catalytic
mechanism of yeast GS. We found that only donor substrates can adopt the
catalytically favorable bent conformation for donor transfer to a growing glycogen
chain.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/12082 |
| Date | 08 December 2016 |
| Creators | Mahalingan, Krishna Kishore |
| Contributors | Hurley, Thomas D., Elmendorf, Jeffrey, Georgiadis, Millie M., Roach, Peter J. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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