An IAA Oxidase-Inhibitor System in Bean Pods. II. Kinetic Studies of Oxidase & Natural Inhibitor

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Abstract
Kentucky Wonder pole bean endocarp and exocarp both contain the enzyme, but only exocarp contains the inhibitor. Endocarp senesces much faster than exocarp unless auxin is added, indicating that the enzyme-inhibitor system may function in the regulation of the onset of senescence. Investigation of the mode of action of the oxidase and the inhibitor revealed the following: (1) After the inhibitor-induced lag is ended the kinetics are identical to those of an inhibitor-free reaction. (2) 2,4-Dichlorophenol (DCP) is needed to end the lag, and the lag is shortened by increasing the amount of enzyme or substrate. (3) Added Mn is needed neither to end the lag, nor for the oxidation of indoleacetic acid (IAA) (although Mn catalyzes the latter). (4) Essentially no oxidation of IAA occurs in the absence of DCP, and with low concentrations of DCP, the oxidation of IAA is slower and preceded by a lag period. (5) H2O2, does not catalyze the oxidation of IAA. (6) Inactivation of the inhibitor is permanent as shown by addition of either more IAA or more enzyme. (7) The inactivation of the inhibitor is temperature dependent. (8) A lag occurs when inhibitor is added before IAA, while when added 3 to 5 minutes after the IAA only a brief shoulder results before the rate of IAA destruction is restored; when the latter procedure is followed using substrate saturation there is no effect on the kinetics of IAA destruction. (9) The inhibitor is inactivated prior to the phase of measurable O2 consumption. These and other data support the interpretation that the oxidation of IAA involves an enzyme-mediated free-radical sequence initiated in the presence of DCP acting in a redox capacity, and that the inhibitor acts as a free-radical trap blocking intermediate reactions prior to the phase of O2 consumption.