Spatial relationship between the copper and carbonyl cofactors in the active site of pig plasma amine oxidase.

The ortho-, meta-, and para-trifluoromethylphenylhydrazine inhibitors of porcine plasma amine oxidase were synthesized. Titrations of plasma amine oxidase with these inhibitors demonstrated that 1 mol of trifluoromethylphenylhydrazine completely and irreversibly modifies 1 mol of enzyme by covalently binding to the active carbonyl cofactor. NMR relaxation measurements on the fluorine nuclei were obtained at 188.22 and 74.84 MHz for each inhibitor-enzyme adduct. These measurements were used to calculate the exact distance and orientation between the inhibitor-binding site and the copper cofactor. The copper lies in the plane of the aromatic ring of the inhibitor at distances of 10.9, 14.3, and 15.5 A from the fluorines in the ortho-, meta-, and para- positions of the ring, respectively. Since the inhibitors react with the active carbonyl cofactor, this defines the relationship between the copper and the active carbonyl cofactor in the enzyme, and provides a basis for choosing between mechanisms for the transfer of electrons from the amine substrate to oxygen.