Abstract“What I cannot create, I do not understand” – Richard Feynman
While these famous words may stem from a physicist, they appear to have inspired scientists from a wider range of backgrounds: one of the key objectives of synthetic biologists is the design of functional proteins, a field which is said to have recently ‘come of age’ (1). The maquette approach provides a rational way of designing proteins without the complexity of their natural counterparts. The Anderson group have employed this method to produce a variety of four-helix bundle proteins, including C45, a heme-binding peroxidase with impressive thermostability (2).
This work focusses on the structural and functional impact of organic cosolvents on C45. The secondary structure was analysed by circular dichroism in concentrations of 10-80% acetonitrile, methanol, ethanol, isopropanol and TFE. A slight increase in helicity could be observed in up to 60% of any of the alcoholic cosolvents. In high (up to 80%) TFE concentrations, this was particularly pronounced, confirming the ability of the cosolvent to stabilise α-helices (3–5). NMR spectroscopy also suggested that TFE may have a stabilising effect on the enzyme.
Further, the effect of the cosolvents on the kinetics of the reaction between C45, ABTS and H2O2 was investigated under limiting peroxide concentration. While the general trend observed was a decrease in kinetic activity, in TFE, the activity increased. In the highest tested cosolvent concentration of 80% TFE, the kcat showed a 7-fold increase compared to that in buffer. Total turnover numbers increased even more significantly, by a factor of 34. This was highly unusual. A smaller increase in
kinetic activity upon the addition of TFE is not unprecedented (6), however not commonly in concentrations above 30%.
This was investigated further using stopped flow spectrophotometry. The reaction did not reach saturation and no kcat or KM for H2O2 could be determined, however an estimate suggests the kcat/KMa/KMb increased 4-fold compared to the reaction in buffer. Further, it was observed that both in buffer and 80% TFE, v0/[E]0 increased significantly with increasing enzyme concentration. This very unusual observation was attributed to different levels of catalytic activity present in the dimer and monomer of C45. The presence of both of those was confirmed by analytical SEC as well as SEC-SAXS.
The mechanism of the formation of compound I, the radical intermediate in the catalytic cycle of heme peroxidases, was investigated using stopped-flow spectrophotometry of the reaction between C45 and H2O2. First, the kinetic isotope effect was exploited to confirm which phase of this reaction corresponded to compound I formation. This process was shown to be significantly faster in 80% TFE than buffer. This leads to the conclusion that stabilisation of the radical intermediate compound I plays an important role in the TFE-mediated increase in catalytic activity of C45.
This work could provide a route to improving the catalytic activity of small heme peroxidases, which may lay the foundation of novel applications of maquette proteins, especially with the possibilities offered by the rapidly growing research field of enzyme engineering. This is especially relevant in light of the industrial interest in non-aqueous enzymology (7).
|Date of Award||29 Sep 2020|
|Supervisor||David J Fermin (Supervisor) & J L R Anderson (Supervisor)|
- Peroxidase, C45, Enzyme, Kinetics, non-aqueous enzymology, Enzymology, Solvents, TFE, Trifluoroethanol, Michaelis-Menten kinetics, Ping-pong kinetics, Protein structure, Circular Dichroism, de novo enzyme