A study of enzymatic methyl transfer catalysed by COMT: mechanism and structural biology

UoM administered thesis: Phd

  • Authors:
  • Sylwia Czarnota

Abstract

Looking at protein structure and studying changes in active site geometry is a fundamental step to understand how enzymes work, how the reaction proceeds and it gives absolutely essential background for potential drug design and development. Enzymes that transfer methyl groups (methyltransferases, MTs) are exciting targets for therapeutic intervention in a range of disorders. COMT (catechol-O-methyltransferase) is a model system for the study of enzyme-catalysed methyl transfer, but is also a very important drug target, as inhibition of this enzyme is a strategy for the treatment of a range of neurological disorders including Parkinson’s disease, depression and schizophrenia. This thesis primarily concerns the use of nuclear magnetic resonance (NMR) spectroscopy to study the reactant and transition state analogue (TSA) of human S-COMT. To achieve that, firstly, two NMR backbone assignments of COMT ternary complexes were determined. One with sinefungin, a fungal-derived inhibitor that possesses transition state-like charge on the transferring methyl group; and the second with S-Adenosyl-L-methionine (SAM), which is the major methyl donor for MTs, naturally present in organisms. Two X-ray crystal structures of the same complexes were obtained in high resolution. Comparisons between these complexes were done with the aid of computational studies, identifying subtle conformational differences in the active sites of the two ternary complexes. Results were consistent between all three methods, leading to the conclusion of active site “compaction” and electrostatic stabilization between the transferring methyl group and “equatorial” residues that are orthogonal to the donor-acceptor coordinate. High pressure NMR (up to 2500 bar) was next used to probe protein flexibility and rigidity, as well as NMR relaxation measurements, to study dynamics of the backbone. Both indicated high stability of the protein and showed that the majority of the protein is highly ordered. Those methods also indicated C-terminus stabilisation, most likely due to the dimer interface occurring there, which could be the focus of future work.

Details

Original languageEnglish
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Award date1 Aug 2019