The Order of Substrate Addition and Product Release during the Catalytic Action of Pig‐Plasma Benzylamine Oxidase

1 Hydrogen peroxide is neither released, nor catalytically formed, during the anaerobic reduction of pig‐plasma benzylamine oxidase by the substrate benzylamine. This observation rules out the possibility that oxygen remains tightly bound (in a catalytically relevant manner) to the enzyme under standard experimental conditions of anaerobiosis, i.e. the possibility that benzylamine oxidase operates by a ternary‐complex mechanism is definitely excluded. 2 During benzylamine oxidase catalysis in the presence of oxygen there is a transient burst of hydrogen peroxide release preceding the steady‐state phase of product formation. It can be inferred from the variation with pH and oxygen concentration of the rate and amplitude of this burst that hydrogen peroxide formation is associated with the oxygen‐dependent reaction step in the process of enzyme reoxidation. Approximately one mole of hydrogen peroxide per mole of enzyme is released in the burst observed at high pH and oxygen concentration, confirming that benzylamine oxidase contains a single catalytically active site. 3 There is no production of ammonia from benzylamine during the catalytic action of benzylamine oxidase under anaerobic conditions. This observation rules out recently proposed mechanisms of enzyme action based on the presence of a sulfenic acid residue or a disulfide group functioning as an electron acceptor at the active site of the enzyme. It lends support to the original suggestion that benzylamine oxidase is a pyridoxal enzyme.