Biodegradative Threonine Dehydratase. Reduction of Ferricyanide by an Intermediate of the Enzyme-Catalyzed Reaction

Abstract

The threonine-dependent reduction of ferricyanide catalyzed by the purified biodegradative threonine dehydratase of Escherichia coli has been studied. The rate of production of 2-oxobutyrate in the presence of ferricyanide was lower than that found in the absence of ferricyanide. The concentrations of threonine required for half-maximal effects for the reduction of ferricyanide and, in the presence of the dye, for 2-oxobutyrate production, were 3 mM and 9mM, respectively. Reduction of ferricyanide was accompanied by evolution of CO2, and even within a very short incubation time with the enzyme, the ratio of ferricyanide reduced over CO2 evolved was approximately 7. Stopping the enzyme activity after a brief exposure to threonine at pH 9.7 resulted in the accumulation of an intermediate (with a half-life of 4 min at 25 degrees C) which formed an adduct with N-ethylmaleimide; the accumulated intermediate, in the absence of N-ethylmaleimide, reduced ferricyanide with concomitant evolution of CO2. We conclude from these results that 2-aminocrotonate is the intermediate which serves as a source of reducing equivalent for ferricyanide, and nonstoichiometric amount of ferricyanide reduction may be attributed to some secondary reactions of ferricyanide with compounds derived from the oxidation product of 2-aminocrotonate.