Abstract
An allosteric binding site with high affinity for imidazoline I(2) ligands has been proposed to exist on monoamine oxidase-B (MAO-B). However, enzyme inhibition only occurs at ligand concentrations far higher than are required to saturate this site. We here confirm previous reports that inactivation of recombinant human MAO-B with tranylcypromine results in the formation of a high affinity I(2) site on the enzyme, measured as an increase in binding of [(3)H]2-BFI. Incubation of MAO-B with 2-phenylethylamine, an endogenous trace amine and MAO-B substrate, resulted in a progressive loss of enzyme activity, increased enzyme mass, distinct spectral changes and, as was observed with tranylcypromine, a parallel increase in high affinity binding of [(3)H]2-BFI. Kinetic studies of the mechanism by which 2-BFI inhibits MAO-B activity suggested binding of 2-BFI, at micromolar concentrations, to a site distinct from the active site on at least two forms of the pure enzyme, probably corresponding to oxidised and reduced enzyme states. Studies with mutant enzymes revealed a pattern of changes consistent with binding of 2-BFI to the substrate entrance channel of human MAO-B. Structural data confirm that high affinity binding of I(2) ligands occurs within the entrance channel of inactive enzyme, while lower affinity binding at the same location in catalytically active enzyme results in mixed inhibition of MAO-B activity. High affinity I(2) sites may form in vivo due to inactivation of a portion of MAO-B during amine oxidation, while the low affinity I(2) site on active enzyme is a target for novel MAO-B inhibitor drugs.