RT Journal Article
SR Electronic
T1 Glu-370 in the Large Subunit Influences the Substrate Binding, Allosteric, and Heat Stability Properties of Potato ADP-glucose Pyrophosphorylase
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 054106
DO 10.1101/054106
A1 Ayse Bengisu Seferoglu
A1 Seref Gul
A1 Ugur Meric Dikbas
A1 Ibrahim Baris
A1 Kaan Koper
A1 Mahmut Caliskan
A1 Gul Cevahir
A1 Ibrahim Kavakli
YR 2016
UL http://biorxiv.org/content/early/2016/05/18/054106.abstract
AB ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWT AGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of the AGPase.AbbreviationsAGPaseADP-glucose pyrophosphorylaseBSAbovine serum albuminDTTdithiothreitolG1Pglucose-1-phosphateIPTGisopropyl-P-D-thiogalactopyranosideLSlarge subunit3PGA3-phosphoglyceric acidPiinorganic phosphateSSsmall subunitTBSTris-buffered salineWTwild type