Steady-State Kinetic Mechanism of Recombinant Avocado ACC Oxidase: Initial Velocity and Inhibitor Studies†

The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The Kd value for Fe(II) (40 nM, free species) and the Km's for ascorbic acid (2.1 mM), ACC (62 μM), and O2 (4 μM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the...