Structural and functional studies of phosphoenolpyruvate carboxylase kinase

Lennie, Gregor Robert (2010) Structural and functional studies of phosphoenolpyruvate carboxylase kinase. MSc(R) thesis, University of Glasgow.

Abstract

Phosphoenolpyruvate carboxylase (PEPc) catalyses the irreversible β-carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and inorganic phosphate. PEPc plays an anaplerotic role in all plants and bacteria, providing citric acid cycle intermediates. The enzyme also catalyses the primary carboxylation step in C4 photosynthesis and Crassulacean Acid Metabolism (CAM), and has more specialised roles in guard cell opening and fruit ripening. Due to the importance of the role of PEPc, the enzyme must be tightly controlled. This is achieved in two ways: allosteric regulation and covalent modification. The enzyme can be activated by glucose 6-phosphate and inhibited by malate. Superimposed on this, phosphorylation of PEPc alters the allosteric properties, making it more susceptible to activation and less susceptible to inhibition. The enzyme that catalyses phosphorylation of PEPc is PEPc kinase. The initial objective of this project was to solve the structure of PEPc kinase by X-ray crystallography. Crystallisation conditions in the presence and absence of an ATP analogue were investigated using PEPc kinase from soybean expressed heterologously in yeast, but only one "hit" of microcrystals was obtained. Using a fresh batch of protein, optimisation around the conditions that gave microcrystals was unsuccessful. Owing to time constraints, further attempts at cystallisation were abandoned. The use of NMR to solve the structure of the kinase in solution was not practicable owing to the relatively low amounts of kinase that can be obtained. The quaternary structure of the protein was analysed using size exclusion chromatography on Superose 12. This showed that the protein, despite migrating as a single band on an SDS-PAGE gel, was composed of two peaks with different specific activities. The relative intensities of the two peaks were not influenced by temperature or the presence of ATP/Mg. Since the two peaks could be due to oligomerisation, analytical ultracentrifugation analysis of recombinant PEPc kinase was carried out. Sedimentation velocity analysis gave a lower sedimentation coefficient than a homology model of soybean PEPc kinase would suggest. Sedimentation equilibrium analysis showed no evidence for oligomerisation of the protein. Circular dichroism spectroscopy showed the presence of significant amounts of α-helix and β-sheet. Thus it was suggested that the two peaks in size exclusion chromatography arise due to the existence of two different monomeric conformations, one more open than the other. All plants contain a small family of PEPc genes and a small family of PEPc kinase genes, raising the possibility that specific PEPc kinases could target specific PEPcs. The kinetic properties of PEPc kinases from soybean. Sorghum and Arabidopsis were measured using PEPcs from Kalanchoë, maize and rice. The data show that all of the PEPc kinases can use all of the PEPc isoforms, arguing against kinetically determined specific PEPc/PEPc kinase pairs. It has recently been discovered that an isoform of PEPc is targeted to the chloroplast of rice. This isoform contains the consensus phosphorylation sequence for PEPc kinase, which raises the question of whether this protein is a substrate for the known rice PEPc kinases. The rice PEPc kinases were expressed in vitro and used in a kinase assay with rice chloroplast PEPc as substrate. This confirmed that rice PEPc kinases can phosphorylate rice chloroplast PEPc in vitro.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Life Sciences
• Supervisor's Name: Nimmo, Professor Hugh
• Date of Award: 2010
• Depositing User: Mrs Marie Cairney
• Unique ID: glathesis:2010-82403
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 25 Aug 2021 10:17
• Last Modified: 25 Aug 2021 10:17
• Thesis DOI: 10.5525/gla.thesis.82403
• URI: https://theses.gla.ac.uk/id/eprint/82403