Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol-O-methyltransferase

Area of Science:

• Biochemistry and Enzymology
• Structural Biology
• Computational Chemistry

Background:

• Catechol-O-methyltransferase (COMT) is a key enzyme in neurotransmitter deactivation, utilizing S-adenosyl-l-methionine (SAM).
• The precise catalytic mechanism and active site dynamics of COMT remain incompletely understood.
• Experimental data probing active site geometry, dynamics, and electrostatics are crucial for elucidating COMT's function.

Purpose of the Study:

• To investigate the physical basis of catalysis in COMT using a transition state analog approach.
• To determine the structural and dynamic features of COMT in complex with substrates and inhibitors.
• To elucidate the role of active site electrostatics and geometry in facilitating methyl transfer.

Main Methods:

• Utilized sinefungin as a transition state analog for COMT in combination with a catechol substrate.
• Determined X-ray crystal structures of ternary complexes involving human COMT, dinitrocatechol, Mg2+, and either SAM or sinefungin.
• Performed NMR backbone assignments and employed density functional theory (DFT) calculations and molecular dynamics (MD) simulations.

Main Results:

• Established sinefungin as a viable transition state analog for COMT.
• Observed evidence of active site 'compaction' driven by electrostatic stabilization between the methyl group and orthogonal active site residues.
• Demonstrated that the enzyme is preorganized upon substrate binding, with catalysis facilitated by electrostatic interactions orthogonal to the reaction coordinate.

Conclusions:

• COMT catalysis is significantly influenced by electrostatic stabilization of developing charge via interactions orthogonal to the primary reaction coordinate.
• The enzyme exhibits geometric preorganization, minimizing conformational changes required for methyl transfer after substrate binding.
• Active site compaction and electrostatic interactions play critical roles in the efficient deactivation of catecholamine neurotransmitters by COMT.