Investigating the contribution of protein dynamics to catalysis in protochlorophyllide oxidoreductase

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Enzyme dynamics has been established to play a crucial role in catalysis, and it has therefore become an important area of research to better understand enzymatic rate enhancements. The light-activated enzyme protochlorophyllide oxidoreductase (POR) is a well-studied model system where dynamics are known to be important for catalysis. The catalytic reaction involves a sequential hydride and proton transfer to reduce the C17-C18 double bond in the protochlorophyllide (Pchlide) substrate with NADPH as a cofactor to yield the chlorophyllide (Chlide) product. Both H-transfer steps are established to undergo quantum tunneling, as derived from the temperature-dependence of the kinetic isotope effects (KIEs). Furthermore, a role for ?promoting motions/vibrations? has been presumed from the temperature-dependence KIE data, which will be investigated further in this thesis by the study of the KIE response to pressure. A general overview of the pressure-dependence as a new experimental probe is presented and compared with temperature-dependencies of KIEs, to establish whether pressure is suitable as an alternative technique for studying the role of enzyme dynamics in catalysis. This involves a comparison of pressure data from other enzyme systems to newly collected data for POR. However, no clear trend between temperature and pressure data is observed and hence, it can be concluded that pressure effects can be difficult to interpret. A case by case analysis is required and needs to be combined with computational simulations based on structural evidence (e.g. X-ray crystallographic), which is not yet available for POR.Solvent-viscosity has been successfully used to probe enzyme dynamics in POR and provides information on the extent of any protein networks that are involved along the reaction coordinate. Here I investigate the solvent-viscosity dependence of both H-transfer reactions in POR for a range of homologous POR enzymes to obtain an evolutionary perspective of the protein dynamics required for catalysis. This has been successfully used in the past on a limited number of POR homologues and has led to the formulation of a hypothesis supporting a twin-track evolution of the two catalytic steps in POR. I observed a lack of solvent-viscosity dependence in case of the hydride transfer across all the investigated lineages, while the proton transfer was shown to be more strongly affected by viscosity in prokaryotic enzymes than in their eukaryotic counterparts. This supports the proposed theory, suggesting an early optimisation of the dynamics involved in the light-activated hydride transfer with a strong reliance on localised motion. Conversely, the proton transfer experienced selective pressure to reduce its dependence on complex solvent-slaved motion and that has led to localised dynamics in eukaryotic POR homologues. Additionally, I found that the enzymes from eukaryotic species have a higher rate of both H-transfer steps, suggesting that an optimisation of the active site architecture occurred upon endosymbiosis. Enzyme dynamics clearly have a pivotal role to play in catalysis of this unique light-activated enzyme and detection of these will only be possible by detailed structural information.