Development of new tools for the characterisation of transient enzyme species by EPR spectroscopy

  • Emma Castiglioni

Student thesis: Phd


By using light as trigger it is possible to study many biological processes, such as the activity of genes, proteins and other molecules, with precise spatiotemporal control. Caged compounds, where biologically active molecules are generated from an inert precursor upon photolysis, offer the potential to initiate such biological reactions with high temporal resolution [1 to 3]. As light acts as the trigger for cleaving the protecting group the ‘caging’ technique provides a number of advantages as it can be intracellular, rapid and controlled in a quantitative manner [1 to 5]. In general, to design suitable caged compounds two important requirements are: 1) the uncaging step needs to be faster than the process under study; 2) the efficiency of uncaging should be high to remove easily the caging groups. Another main point to consider is that caged compounds must be biologically inert before photolysis [2, 3 and 5]. A caging strategy was applied to study the catalytic cycle of ethanolamine ammonia lyase (EAL). EAL is a coenzyme B12–dependent enzyme and it has become an important model system to understand the catalytic mechanism of cobalamin B12-dependent enzymes in general. In particular, it has been used to study the relationship between catalysis and enzyme–substrate complex geometry, and the properties of the active site [6]. Moreover, in conjunction with TgK Scientific, a novel rapid freeze-quench (RFQ) instrument, which combines fast mixing and flashing capabilities, was characterised. The purpose of this device consists in trapping reaction intermediates at low temperatures and analysing them by electron paramagnetic resonance (EPR) spectroscopy to identify the involvement of any radical species during catalysis [7 and 8].
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorNigel Scrutton (Supervisor), Stephen Rigby (Supervisor) & Alistair Fielding (Supervisor)


  • EPR
  • photolysis
  • caged-compounds
  • freeze-quench

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