Equatorial active site compaction and electrostatic reorganization in catechol-O-methyltransferase

Sylwia Czarnota, Linus O. Johannissen, Nicola Jane Baxter, Felix Rummel, Alex Wilson, Matthew John Cliff, Colin W. Levy, Nigel S. Scrutton, Jonathan Peter Waltho, Sam Hay

Research output: Contribution to journalArticlepeer-review


Catechol-O-methyltransferase (COMT) is a model S-adenosyl-L-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate. Here we establish that sinefungin, a fungalderived inhibitor of SAM-dependent enzymes that possess transition state-like charge on the transferring group, can be used as a transition state analog of COMT when combined with a catechol. X-ray crystal structures and NMR backbone assignments of the ternary complexes of the soluble form of human COMT containing dinitrocatechol, Mg2+ and SAM or sinefungin were determined. Comparison and further analysis with the aid of density functional theory calculations and molecular dynamics simulations provides evidence for active site ‘compaction’, which is driven by electrostatic stabilization between the transferring methyl group and ‘equatorial’ active site residues that are orthogonal to the donor–acceptor (pseudo reaction) coordinate. We propose that upon catecholamine binding and subsequent proton transfer to Lys 144, the enzyme becomes geometrically preorganized, with little further movement along the donor–acceptor coordinate required for methyl transfer. Catalysis is then largely facilitated through stabilization of the developing charge on the transferring methyl group via ‘equatorial’ H-bonding and electrostatic interactions orthogonal to the donor–acceptor coordinate.
Original languageEnglish
Pages (from-to)4394-4401
Number of pages8
JournalACS Catalysis
Issue number5
Early online date9 Apr 2019
Publication statusPublished - 2019


  • NMR
  • S-adenosyl- l -methionine
  • X-ray crystallography
  • density functional theory
  • enzyme
  • molecular dynamics simulation
  • sinefungin


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