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Title: Phosphorylation of C4 and non-C4 phosphoenolpyruvate carboxylase from panicum and the kinetic behaviour of phosphorylated and non-phosphorylated PEPC
Author: Zawawi, Dhiya Dalila
ISNI:       0000 0004 9356 9211
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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The C4 photosynthesis pathway is more efficient than C3 photosynthesis due to the capability of phosphoenolpyruvate carboxylase (PEPC) that provides a CO2 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, a 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 SDS-PAGE gel. The phosphate affinity Phos-TagTM was performed subsequently to ensure all PEPCs present were fully phosphorylated. Peptides resulting from the trypsin digestion of Phos-TagTM SDS-PAGE gels were analysed by mass spectrometry to identify phosphorylation site on PEPC. Two phosphopeptides were detected in the PEPC from P. queenslandicum and six from the 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 nonphosphorylated 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 nonphosphorylated 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 protects the PEPCs against malate and aspartate inhibition.
Supervisor: Reid, James Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available