The mechanism of inhibition of pig-plasma benzylamine oxidase by the copper-chelating reagent cuprizone.

Abstract

Treatment of pig‐plasma benzylamine oxidase with the copper‐chelating reagent cuprizone leads to the reversible formation of an enzyme · cuprizone complex with a dissociation constant of about 3 mM, followed by an irreversible conversion of this complex into an enzyme species exhibiting a new absorption band centered around 350 nm. The latter enzyme species shows no reactivity towards substrate or carbonyl reagents such as phenylhydrazine. One mole cuprizone per mole protein is sufficient to complete the chromophoric changes and to inactive the enzyme completely. Cuprizone reacts with free pyridoxal phosphate under formation of a reaction product showing maximum absorption at 350 nm in the visible region, and competes with phenylhydrazine for the protein‐bound pyridoxal phosphate in benzylamine oxidase. Cuprizone does not extract any significant amounts of copper from benzylamine oxidase, but affects electron paramagnetic resonance spectra of the enzyme and its phenylhydrazone derivative in a way suggesting a direct interaction with at least one of the two copper ions present in the protein. The latter interaction does not appear to be identical with those involved in the formation of the inactive enzyme species absorbing at 350 nm. It is concluded that the inhibitory action of cuprizone cannot be attributed to its copper‐chelating properties, but reflects an interaction with enzyme‐bound pyridoxal phosphate through a mechanism analogous to that governing Schiff‐base or hydrazone formation between enzyme and substrates or hydrazine derivatives.