Catechol-O-methyltransferase (COMT): Resolving the mechanism of an archetypical methyl transferase with new experimental tools

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The primary means of deactivation of catecholamine neutrotransmeters such as dopamine, levodopa and (nor)epinephrine occurs through the action of an enzyme called catechol-O-methyltransferase (COMT). Consequently, inhibition of COMT is a strategy for the treatment of a range of neurological disorders including Parkinson's disease, depression and schizophrenia. In addition to being a drug target, COMT has potential as a useful biocatalyst to create valuable chemicals such as vanillin, the primary component of the extract of the vanilla bean. Clearly a detailed understanding of how COMT works would be of value to a range of sectors. However, despite more than 60 years of study, there are still major disagreements as to fundamental aspects of the physical mechanism of the reactions catalysed by methyl transferases such as COMT. The focus of this work is to study COMT using new experimental tools - NMR and optical spectroscopies, X-ray crystallography - alongside a tightly integrated computational chemistry programme, with a focus on the physical mechanism of catalysis by this enzyme. This work will capitalise on our recent work, which produced the first NMR backbone assignments of COMT, which is necessary to map NMR data onto new and/or existing X-ray crystal structure data. We will extend this approach to study how COMT binds a range of physiological substrates such as dopamine. By using our NMR experiments as a screening method, we expect to optimise sample conditions so we can solve the first X-ray crystal structures of COMT with bound catecholamines, allowing better description of neurotransmitter binding to COMT. We will expand on our previous NMR work to perform more technically demanding high pressure and relaxation NMR experiments that directly probe protein flexibility and dynamics. Alongside optical fluorescence and vibrational experiments, these experiments will allow the identification of any specific dynamic features - amino acid backbone and/or side chain motions - that may contribute to catalysis. The significance of such behaviour will be investigated by altering amino acids of interesting (by site-directed mutagenesis) and by comparing the activity (enzyme turnover) of wild-type and mutant enzymes lacking amino acids of interest. This approach allows the direct assessment of dynamical features to COMT catalysis - a major area of controversy in the wider enzymology community.A major feature of the proposal is the close integration of computational simulations with experimental work, which will be performed both in Manchester and by collaborators at the University of Arizona. Experiments are designed to provide data that can be directly used as inputs to computational calculations. Calculations and simulations will be directly tested/benchmarked against experimental data to allow successive improvements to computational parameters and to guide the design of subsequent experiments, e.g. in the identification of amino acids that may play a major role in catalysis. Together, this approach will allow new insight into how COMT - and by extension, related enzymes - works. In addition to resolving long-standing questions in the field, this work could lead to improvements in drugs designed to inhibit COMT and related enzymes, will provide foundational data that will aid in the rational (re)design of related methyl transfer enzymes for use in biocatalysis applications, and will provide new methodology to tackle similar mechanistic questions in a wide range of other enzymes.