Catalysis at the Interface- Elucidation of the Activation Process and Coupling of Catalysis and Compartmentalization of the Peripheral Membrane Protein Pyruvate Oxidase from Escherichia coli

Catalysis at the Interface- Elucidation of the Activation Process and Coupling of Catalysis and Compartmentalization of the Peripheral Membrane Protein Pyruvate Oxidase from Escherichia coli

Sitte, Astrid

Dissertation

Angenommen am: 2013-04-24

Erschienen: 2014-02-06

Betreuer: Tittmann, Kai Prof. Dr.

Gutachter: Tittmann, Kai Prof. Dr.

Gutachter: Diederichsen, Ulf Prof. Dr.

Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0022-5E14-3

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Name: PhD Thesis_Astrid Sitte.pdf

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Beschreibung: Dissertation

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Zusammenfassung

Englisch

The thiamine- and flavin-dependent peripheral membrane protein pyruvate oxidase from E. coli (EcPOX) catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO2 and acetate with a concomitant electron transfer step to ubiquinone 8 (Q8), a membrane-bound mobile carrier of the respiratory chain. Association of EcPOX to the lipid bilayer is conferred by the last 23 amino acids of the C-terminal part which form an amphipathic helix in the presence of membranes. Binding to the lipid bilayer is not only mandatory for feeding the reducing equivalents into the electron transport chain, but also leads to enhancement of the catalytic activity of the enzyme. In this thesis the activation and membrane binding mechanism of EcPOX was investigated. EcPOX was used as well-suited model system to analyze the impact of multiple amino acids on an enzymes activation mechanism and eventual bilayer association. Intensive kinetic and thermodynamic studies were performed in combination with X-ray crystallography. The results allowed the creation of a molecular activation model and give insights into a close coupling mechanism of catalysis and compartmentalization of an occasionally active enzyme. It was shown that amino acids within the active site mediate pyruvate-induced reduction of the protein-bound flavin cofactor. The flavin redox state is also sensed by specific amino acids. Thus, the flavin acts as redox switch and a signal transfer cascade starting from the reduced flavin leads to exposure of the hitherto protein-bound membrane anchor. This membrane anchor is attached to the protein surface by hydrophobic interactions with the protein core. Upon activation these contacts are disturbed by conformational changes of the protein and the lipid binding domain to ensure membrane anchor exposure. Concomitant to release of the membrane anchor also an autoinhibitory helix is removed from the protein which leads to a great enhancement of the catalytic activity of EcPOX. Finally, the membrane anchor undergoes a last conformational transition in the presence of membranes. Bilayer association of the enzyme is thereby promoted by the formation of an amphipathic helix from the membrane anchor. Similar to the stabilization at the protein the membrane anchor is also bound to the membrane by hydrophobic contacts. Attached to the bilayer EcPOX has then access to its substrate Q8 and can complete its catalytic cycle. Initial results suggest that Q8 leaves the membrane and penetrates the active site of EcPOX according to an out-of-the membrane mechanism.

Keywords: EcPOX; thiamine diphosphate; FAD; membrane binding; limited proteolysis; activation; ubiquinone 8; electron transfer; amphipathic helix; X-ray structure; out-of-the-membrane mechanism; conformational change; autoinhibition