L-Threonine dehydrogenase of Escherichia coli K-12.

A rapid method for purifying L-threonine dehydrogenase from Escherichia coli K-12 cells, grown on a medium containing L_-threonine as carbon source, has been developed. The procedure consists of three fractionation steps of which the last is adsorption and elution of the enzyme from a column of Blue dextran-Sepharose. Homogeneous and stable samples of the dehydrogenase are obtained. L-Threonine dehydrogenase of E. coli has an average molecular weight of 140,000 and consists of 4 identical or nearly identical subunits. Km values for L--threonine and NAD @ are 1.4 mM and 0.19 mM_, respectively; several substrate and coenzyme analogs are also active. The pH optimum is 10.3 and Mn ++ stimulates dehydrogenase activity. Other properties of the pure enzyme have been established. INTRODUCTION Three pathways are currently known whereby L_-thre0nine, an essential amino acid for mammals, is catabolized. In one, the cleavage of threonine into acetaldehyde plus glycine, as catalyzed by threonine aldolase, is the first reaction. Threonine dehydratase initiates another route wherein threonine is converted to ammonia and 2-ketobutyrate; the latter compound, in turn, is metabolized to propionyl CoA. The third pathway, in which threonine dehydrogenase is the first enzyme, has received considerably less attention but appear to be assuming a position of greater importance in the metabolism of threonine by both eucaryotes and procaryotes. Of the three enzymes that initiate these separate routes for threonine degradation, only threonine dehydrogenase has resisted extensive purification; the properties of the pure enzyme have not been reported so far and numerous facets of the complete pathway in which it catalyzes the first step [L-threonine + NAD 8 + (~-amino-8-ketobutyrate) + aminoacetone + CO 2 + NADH] have not been delineated. Elliott (i) first observed that aminoacetone accumulated as a product when washed cells of