A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine β-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans-glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1—5mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine—fumarate conjugate, N-(2-phenylethyl)fumaramide (PEA-Fum), is an 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine α-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine β-mono-oxygenase.
Improvement of the Gaussian Electrostatic Model by Separate Fitting of Coulomb and Exchange-Repulsion Densities and Implementation of a new Dispersion term
