Vibrational control of enzymes as a tool for enzyme engineering

Project Summary
The vast majority of biological reactions are catalysed by enzymes, which often exhibit exquisite selectivity and massive catalytic rate enhancements. In many cases, it would be desirable to have a rapid and real-time method of tuning enzyme activity, e.g. to control flux through a multi-enzyme cascade or in vivo metabolic pathway. One possibility is through direct vibrational control of the enzyme reaction, where IR light is used to excite vibrations that couple to the reaction coordinate. It is now becoming practical to directly excite molecular vibrations using ultrafast (sub-picosecond) pulses of infrared (IR) light, which can lead to significant rate enhancements of thermal reactions in the gas-phase and on surfaces, without excessive heating.
Enzymes represent a unique environment for vibrational control, but this has yet to be demonstrated in the literature. Their large size results in many vibrational modes that can couple to substrate and cofactor vibrations within the active site, leading to efficient vibrational relaxation. However, they often possess highly preorganised active sites that hold reactants in a reactive configuration that requires minimal structural reorganisation (after substrate binding) to adopt a transition state geometry. This confinement and steric control is expected to increase the quantum yield of any vibrationally-excited reaction and any observable IR control will provide unique information on the enzyme active site; information that would be valuable for rational enzyme engineering and drug design.
Recently, we have demonstrated that in combination with specific isotopic labelling, it is possible to use IR excitation to alter the rate of an enzyme-catalysed reaction; i.e. via vibrational control. However, we have only demonstrated this IR control in one enzyme, so the main objective of this project is to explore IR control of a wider range of enzyme-catalysed reactions, and to explore applications of this technique within biocatalysis and enzyme engineering. Making use of our established methods and instrumentation, we will initially screen a range of enzyme reactions where H-transfer is rate limiting. These experiments will make use of deuterated coenzymes and substrates, which are commercially available and/or prepared in-house. In the longer-term, we will explore other classes of reactions, with a focus on enzymes that are established in biocatalysis, and the incorporation of IR probes/vibrational control into rational enzyme engineering programmes.
This work is highly interdisciplinary and will involve (i) instrument and method development alongside new data analysis and experimental design procedures, (ii) mechanistic enzymology/ biocatalysis, with an emphasis on developing new analysis that can feed into enzyme engineering, (iii) new and established synthetic methods for the synthesis of isotopically labelled compounds (substrates and cofactors), and (iv) computational chemistry (MD simulations and/or DFT modelling) that will be used to aid in experimental design and interpretation of results.
Overall, the work will provide a highly interdisciplinary approach to biocatalysis, chemistry and biophysics-based research, and falls within the iCAT remit of technology-driven solutions to ICAT system design and reaction monitoring in biosystems. It will offer highly diverse training opportunities to a PhD student, who will have the additional benefit of being able to access the supervisors' laboratories on a daily basis, as they are co-located within the Manchester Institute of Biotechnology (www.mib.ac.uk).