Phosphorylation of C4 and Non-C4 phosphoenolpyruvate carboxylase from panicum and the kinetic behaviour of phosphorylated and non-phosphorylated PEPC

The C4 photosynthesis pathway is more efficient than C3 photosynthesis due to the capability of phosphoenolpyruvate carboxylase (PEPC) that provides a C02 high concentration at Rubisco, thus reducing the photorespiration rate. PEPC is regulated by internal metabolite with malate or aspartate as its...

Full description

Saved in:
Bibliographic Details
Main Author: Dhiya Dalila Zawawi (Author)
Other Authors: x
Format: Thesis Book
Language:English
Subjects:
x
x
Tags: Add Tag
No Tags, Be the first to tag this record!
LEADER 04418cam a2200373 7i4500
001 0000100006
005 20210713090000.0
007 axx
008 210310s2019 my eng
020 |a x  
040 |a x 
082 0 0 |a x 
090 0 0 |a on order  
100 0 |a Dhiya Dalila Zawawi   |e author  
245 0 0 |a Phosphorylation of C4 and Non-C4 phosphoenolpyruvate carboxylase from panicum and the kinetic behaviour of phosphorylated and non-phosphorylated PEPC   |c Dhiya Dalila Zawawi. 
246 0 |a x. 
264 0 |c 2019. 
300 |a ix, 216 leaves;   |c 31 cm. 
336 |a text  |2 rdacontent 
337 |a unmediated  |2 rdamedia 
338 |a volume  |2 rdacarrier 
347 |a x 
500 |a x 
502 |a Thesis (Degree of Doctor of Philosophy) - University of Sheffield, 2020 
504 |a Includes bibliographical references (leaves 139-157) 
505 0 |a 1. Introduction -- 2. Material and methods -- 3. Pepck protein expression, purification and application of nusa tag -- 4. Phosphorylation pf pepc with pepck or protein kinase (pka) -- 5. Kinetic behaviour of phosphorylated and non-phosphorylated pepc -- 6. General discussion nad conclusion 
520 |a The C4 photosynthesis pathway is more efficient than C3 photosynthesis due to the capability of phosphoenolpyruvate carboxylase (PEPC) that provides a C02 high concentration at Rubisco, thus reducing the photorespiration rate. PEPC is regulated by internal metabolite with malate or aspartate as its inhibitors, and glucose-6-phosphate (G-6-P) as its activator. Besides, PEPC also regulated by reversible phosphorylation by protein kinase known as phosphoenolpyruvate carboxylase kinase (PEPCK), that leads to an activation of the enzyme by G-6-P and decrease the sensitivity to malate or aspartate. PEPCK shows a high specificity towards PEPC and the reaction has been reported to be light controlled, but the details of mechanisms of PEPC phosphorylation are still unknown. In this study, comparative analysis was performed between phosphorylated PEPCs from C3 Panicum pygmaeum and C4 Panicum queenslandicum produced either by PEPCK or Protein Kinase A (PKA). Over-expression of PEPCK with solubility tag NusA had produces soluble protein but in small amounts, and was insufficient for further analysis. Purifying PEPCK without the NusA tag, was unsuccessful because it exists as an insoluble protein. Thus, PKA was applied since it is known to phosphorylate PEPC. The phosphorylation of PEPC by PKA has been confirmed with fluorescence detection, by combining Pro-Q Diamond and sYPRO Ruby gel stain in 50S-PAGE gel. The phosphate affinity Phos-Tag" was performed subsequently to ensure all PEPCs present were fully phosphorylated. Peptides resulting from the trypsin digestion of Phos-Tag" 50S-PAGE gels were analysed by mass spectrometry to identify phosphorylation site on PEPC. Two phosphopeptides were detected in the PEPC from P. queenslandtcurm eee-so: from tire P. pygmaeum enzyme. Phosphorylation of PEPC changed the pattern of kinetic enzyme activity, as well as the malate and aspartate sensitivity when compared to the non phosphorylated form. The enzyme activity (Vmax) of PEPC from the C4 species P. queenslandicum increased once phosphorylated, but this was not observed in the PEPC from the C3 species P. Pygmaeum. In terms of PEP Km, the phosphorylated P. queenslandicum PEPC, had a lower Km value when compared to the non phosphorylated one. In the P. Pygmeaum PEPC, phosphorylation did not change the Km (PEP) value or the specific activity. Phosphorylation increased the specificity of PEPC to bicarbonate in the enzymes from both P. queenslandicum and P. pygmaeum at pH 8. Phosphorylated PEPC from P. queenslandicum becomes less sensitive to malate and aspartate inhibition at limiting PEP. In P. pygmaeum, phosphorylation of PEPC made it less sensitive to aspartate at limited PEP and to malate at both limited and saturated PEP. Together, these results lead to understanding how the phosphorylation influence the catalytic activity of PEPC in C3 and C4 plant species differently and protect the PEPCs against malate and aspartate inhibition. 
600 0 0 |a x 
610 2 0 |a Universiti of Sheffield --   |x Dissertations  
650 0 |a Dissertations, Academic  
651 0 |a x  
700 0 |a x  
710 2 |a Universiti of Sheffield  
999 |a 1000182124  |b Thesis  |c Reference  |e Tembila Bibliographic & Index Unit