Studies on indoleacetic acid oxidation by liquid medium from crown gall tissue cells: The role of malic acid and related compounds

Abstract

1. 1. Indoleacetic acid oxidation by liquid medium from crown gall tissue culture cells has been studied. The reaction has a pH optimum of 4.5 and requires Mn 2+ and a monohydric phenol. A short lag phase is routinely observed. The appearance of peroxidase and indoleacetic acid oxidising activity in the medium of a tissue culture was followed over a 3 week time course. One function of this enzyme may be to prevent the accumulation of excess inhibitory concentrations of indoleacetic acid. 2. 2. Gel filtration of the medium through Sephadex G-25 produced 3 peaks. The high molecular weight fraction contained peroxidase but had almost completely lost the ability to oxidise indoleacetic acid unless recombined with the first low molecular weight peak. Malic and other compounds of related structure also reactivated in the same way as the natural activator. Mn 2+ and a monohydric phenol were required in each case. Neither effect was due to inhibition of endogenous catalase. The second low molecular weight peak inhibited indoleacetic acid oxidation by extending the lag phase. 3. 3. Both substrate activation and inhibition were observed in the presence of low levels of activator but were overcome with increasing amounts of activator. There was a non-linear dependence of reaction rate on enzyme concentration which was not apparent in the presence of 3.6 M urea. 4. 4. H 2O 2 is not an obligatory intermediate although it greatly decreased the lag phase and enhanced the rate of oxidation. Mn 2+ was unnecessary for these effects. Competitive studies indicated that both malic acid and H 2O 2 may act on the same process in the reaction mechanism. 5. 5. A model is put forward, based on the hypothesis that the reaction is autocatalytic with a cyclic mechanism involving unstable intermediates, for the participation of malic acid and Mn 2+ as well as monohydric phenols and H 2O 2 in the enzymic oxidation of indoleacetic acid by oxygen. 6. 6. In addition to malic acid, a wide range of non-phenolic compounds was tested for activating ability. The structural characteristics of activators are discussed.