Use this URL to cite or link to this record in EThOS:
Title: Understanding the catalytic cycle of membrane pyrophosphatases through structural and functional studies
Author: Wilkinson, Craig
ISNI:       0000 0004 6497 3717
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2017
Availability of Full Text:
Access from EThOS:
Access from Institution:
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.
Supervisor: Goldman, Adrian ; Muench, Stephen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available