PT  - JOURNAL ARTICLE
AU  - Ayse Bengisu Seferoglu
AU  - Seref Gul
AU  - Ugur Meric Dikbas
AU  - Ibrahim Baris
AU  - Kaan Koper
AU  - Mahmut Caliskan
AU  - Gul Cevahir
AU  - Ibrahim Kavakli
TI  - Glu-370 in the Large Subunit Influences the Substrate Binding, Allosteric, and Heat Stability Properties of Potato ADP-glucose Pyrophosphorylase
AID  - 10.1101/054106
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 054106
4099  - http://biorxiv.org/content/early/2016/05/18/054106.short
4100  - http://biorxiv.org/content/early/2016/05/18/054106.full
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