Intermediate State Stabilisation in Enzyme Catalysis Structural studies of two serine proteases and a photosynthetic reaction centre

Sammanfattning: Enzymes play a key role in every organism?s life. They catalyse chemical reactions and may also respond to external stimulus. The reoccurring theme of this thesis is how biologically relevant information can be gleaned from X-ray structures of the enzymes. Firstly, the inhibitor resistance of a human trypsin isoenzyme was rationalised by solving the structure of the native enzyme. In the crystal structure, the Arg-193 residue was shown to clash with the P2? residues of the inhibitors, which prevents efficient inhibitor binding because of steric and/or electrostatic reasons. Secondly, structural details of catalytic mechanism of serine proteases were revealed based on the atomic resolution structure of an elastase acyl-enzyme complex. From the 0.95 Å resolution structure it is apparent that the apoprotein and the acyl-enzyme are almost identical structurally and the acyl ester bond is not distorted. The accurate description of the active site geometry and the observation of catalytically important hydrogen bonds aid the identification of the relevant catalytic processes. Thirdly, the lipidic cubic phase structure of the photosynthetic reaction centre from Rhodobacter sphaeroides was investigated and compared with previous detergent based crystal structures. The crystal structure revealed how lipid molecules can mediate protein-protein interaction in a lipid bilayer and how the structure of the reaction centre is affected by the presence of a lipid bilayer. Finally, we characterised the P⁺Q_A^- state of the reaction centre with X-ray crystallography using the lipidic cubic phase grown crystals. These results showed that there are light induced structural changes in the reaction centres illuminated for extended periods that are implicated in the regulation of electron transfer reactions. These structural changes elucidate a mechanism in which the entire cytoplasmic domain subtly changes conformation to achieve charge stabilization. Although serine proteases and photosynthetic reaction centres catalyse widely differing reactions, their functional mechanisms share common features like conformationally mediated intermediate state stabilisation. The results presented here underline the importance of conformational changes during enzymatic catalysis.