Abstract
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 fungal-derived 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.
Highlights
S-Adenosyl-L-methionine (SAM) dependent methyl transferases (MTases) are ubiquitous bisubstrate Mg2+-dependent enzymes found in plants, animals, and microorganisms.Catechol-O-methyltransferase (COMT) is an archetypalMTase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals
We suggest that progress toward a consensus description of catalysis by COMT, and by extension the MTase enzyme family, requires new experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate; at a minimum the reactant state and transition state (TS)
As sinefungin contains partial TS character, the sinefungin complex will represent a reactant pose that is positioned along the reaction coordinate between the ground state and TS and any conformational change/reorganization that occurs between SAM and sinefungin ternary complexes is likely to be relevant to catalysis
Summary
S-Adenosyl-L-methionine (SAM) dependent methyl transferases (MTases) are ubiquitous bisubstrate Mg2+-dependent enzymes found in plants, animals, and microorganisms. (KIE) measurements from the Schowen group in the late 1970s showed an unusually large and inverse CH3/C2H3 KIE of ∼0.8 for the COMT reaction[11] with much smaller KIEs observed on uncatalyzed model methyl transfer reactions.[7] These data, alongside more recent KIE measurements from the Klinman group showing a correlation between kcat/Km and CH3/C3H3 KIEs on the COMT reaction,[12] have been used as evidence for the role of active site compression or “compaction” during the reaction;[7,13] essentially the squeezing together of the reacting methyl donor and acceptor moieties, which promotes the reaction This description is couched within the framework of the “promoting vibration” hypothesis, which has been used to interpret isotope effects on H-transfer reactions.[14−16] The unusual deuterium KIE on the COMT reaction has received much attention from the computational community, with a number of proposals put forward to describe catalysis by COMT. We find evidence for active site compaction and propose that this is driven by H-bonding between the transferring methyl group and “equatorial” active site residues, rather than by “pushing” along the donor−acceptor axis
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