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Understanding the Catalytic Cycle of Membrane Pyrophosphatases Through Structural and Functional Studies

Wilkinson, Craig (2017) Understanding the Catalytic Cycle of Membrane Pyrophosphatases Through Structural and Functional Studies. PhD thesis, University of Leeds.

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Membrane pyrophosphatases (M-PPases) couple pyrophosphate hydrolysis to the translocation of sodium ions/protons, using the resulting ion gradients to drive abiotic stress resistance and in the infectivity of protozoan parasites. I have solved two M-PPase structures in different catalytic states, combining these with previous structures to update the model of the catalytic cycle of M-PPases. These new structures confirm previous findings that substrate binding breaks interactions between K12.50 and D6.43 due to motion of helix 12, leading to a rearrangement of helix 6 and priming the enzyme for hydrolysis. Previously this information was only visible between the structures of two-distinct M-PPases, a H+-PPase and Na+-PPase. The current structures allow for comparisons to be made between structures of the same type of M-PPase. Electrometric data was acquired using the Nanion SURFE2R technique, which showed a proton-pumping signal was generated by the non-hydrolysable inhibitor, imidodiphosphate. This provided sufficient information to update the model of the complete catalytic cycle, favouring the hypothesised Binding change mechanism, in which substrate binding induces a series of conformational changes during which ion pumping occurs first, followed by substrate hydrolysis. Additionally, crystal optimisation techniques improved the resolution of the Pyrobaculum aerophilum M-PPase structure to 3.8, providing an overview of the K+-independent M-PPase. The hydrolytic centre and ion gate regions showed similar coordination to previous structures, with differences seen in the conformation of several outer ring helices, potentially linked to K+-independence. I also carried out mutational studies investigating K12.46 and T12.49, both involved in K+-independence and found that both mutations were required to generate a K+-dependent variant of PaPPase. Overall, this information has improved our understanding of the structure and function of the membrane pyrophosphatases, providing a basis for drug-design programmes targeting protozoan parasites, to which the membrane pyrophosphatases are a vital part of growth and infectivity.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.731512
Depositing User: Dr Craig Wilkinson
Date Deposited: 24 Jan 2018 15:40
Last Modified: 25 Jul 2018 09:56
URI: http://etheses.whiterose.ac.uk/id/eprint/19131

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