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

Abstract

In a previous article (21) I reported the presence in pods of Kentucky Wonder pole beans of an IAA3 oxidase and a natural inhibitor of this enzyme. Endocarp and exocarp both contain the enzyme, but only exocarp has inhibitor. Endocarp senesces much faster than exocarp unless auxin is added, which indicates that the enzyme-inhibitor system may function in the regulation of the onset of senescence. The assessment of the physiological role of the enzyme-inhibitor system in bean pericarp tissues, however, requires additional information on the permeability of endocarp to the inhibitor, and its metabolism in this tissue. The inhibitors of IAA oxidase found in bean pods (21), other legumes (8, 10), and quackgrass (16) (which confer a lag, in the oxidation of IAA) occur as a heat stable, dialyzable fraction in the supernatant of tissue-homogenates after centrifugation. I (21 ) showed that in bean exocarp the inhibitor is associated with cytoplasmic particles, and is released in quantity as a dialyzable material by grinding or boiling of tissue. Numerous investigations of IAA-destroying enzymes from plant tissues indicate that the enzymes are hemoproteins, based on cyanide inhibition or lightreversible inhibition by CO (6, 20, 23, 24). Galston et al. (6) regarded the oxidation of IAA by the pea enzyme as a peroxidative oxidation, coupled with a light-activated flavoprotein system which forms H202 from 02, thus accounting for the oxygen consumption. Kenten (11) and Stutz (23) concluded that the oxidation of IAA by enzymes from waxpod beans and lupine, respectively, is not dependent upon a flavoprotein. Evidence from studies of the destruction of IAA by purified peroxidases, crude plant enzyme preparations, and a non-enzymic system indicates that a freeradical mechanism is involved. This is suggested by the ubiquity of the inhibitory effect of polyphenols (see review by Ray, 1958), which are known to act as antioxidants in free-radical reactions (4, 26). Kenten (11), however, suggested that the inhibition confered by polvphenols may be due to their being more readily oxidized by the enzyme. Maclachlan and Waygood (12,13) interpreted their data from studies of IAA oxidation by both a wheat leaf enzyme and a non-enzymic manganiversene system as support for the hypothesis that the oxidation proceeds via a freeradical reaction mediated by manganic ions, with oxygen consumption a result of reaction between intermediate substrate-radicals and oxygen. In this scheme, the polyphenols would act as free-radical traps, ratlher than compete for the enzyme site as suggested by Kenten (11). It should be pointed out that the concentrations of manganese (10-3-10-1 M) they used are within a range optimal (0.02-0.5 %) for non-enzymic catalysis by heavy metal ions (26). Yamazaki and Souzu (28) also concluded that the oxygen uptake was a result of a reaction between oxidized substrate-radicals and oxygen, during the catalysis of IAA oxidation by turnip peroxidase. Ray (20) demonstrated that the Omphalia enzyme exhibited both IAA oxidase and peroxidase activity, which he attributed to different forms of the same enzyme. A similar conclusion was reached by Stutz (23) for a purified lupine enzyme, namely, that the enzyme possessed both peroxidase and dehydrogenase centers, the latter using a phenolic acceptor during the process of IAA oxidation. Ray interpreted his results of inhibition by polyphenols and light-reversible inhibition by CO as supporting the occurrence of a free-radical sequence. He suggested the possibility of the oxygen uptake being concerned with the reconversion of ferrous peroxidase to the ferric form or a peroxide complex thereof. This paper is part of a study of the reaction of IAA oxidase and an inhibitor in bean pods, which indicates that a free-radical mechanism is involved.