Steady-State Kinetics of Substrate Binding and Iron Release in Tomato ACC Oxidase

Abstract

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO₂ and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO₂, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O₂; ascorbate can bind after O₂ or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.