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 K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) 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 kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by 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 inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.
Brunhuber
N M
NM
Department of Chemistry and Biochemistry and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California 93106, USA.
Mort
J L
JL
Christoffersen
R E
RE
Reich
N O
NO
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001667:Binding, Competitive / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D019770:Lauraceae / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D004791:Enzyme Inhibitors; D010940:Plant Proteins; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase