Steady-state kinetic mechanism of recombinant avocado ACC oxidase: Initialvelocity and inhibitor studies

The gaseous plant hormone ethylene modulates a wide range of biological pro cesses, including fruit ripening. It is synthesized by the ascorbate-depend ent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyz ed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was e xpressed in Esherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized as say for the purified enzyme was developed that takes into account the inher ent complexities of the assay system. Fe(II) and ascorbic acid form a binar y complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K-d value for Fe(II) (40 nM, free species) and the K-m's for ascorbic acid (2.1 mM), ACC (62 mu M), and O-2 (4 mu M) were determined. Fe(II) and ACC exhibit substrate inhibiti on, and a second metal binding site is suggested. Initial velocity measurem ents and inhibitor studies were used to resolve the kinetic mechanism throu gh the final substrate binding step. Fe(II) binding is followed by either a scorbate or ACC binding, with ascorbate being preferred. This is followed b y the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O- 2 are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate in hibition. BCC and ascorbate analogues were used to confirm the kinetic mech anism and to identify important determinants of substrate binding.