Threonine Deaminase from Salmonella typhimurium: I. PURIFICATION AND PROPERTIES

Abstract

Abstract Biosynthetic l-threonine deaminase (l-threonine hydrolyase, deaminating; EC 4.2.1.16) has been purified approximately 250-fold from crude extracts of nutritionally derepressed Salmonella typhimurium. Sedimentation of the enzyme in the ultracentrifuge results in a single, sharp, symmetrical peak in the schlieren pattern. The purification procedure is capable of removing the radioactivity from a crude extract containing label in all the proteins except native threonine deaminase. The deaminase reaction shows homotropic interaction only in the presence of l-isoleucine; high ionic strength or low valine concentrations remove these interactions. The pure enzyme is effectively inhibited by l-isoleucine. The pH optimum of the deaminase reaction is 9 to 10; the pH curve is shifted to higher values in the presence of l-isoleucine. The activity of the purified enzyme is independent of added pyridoxal phosphate. The cofactor is resolved by dialysis against tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 8.0. Pyridoxal phosphate and pyridoxamine phosphate are capable of reactivating the enzyme. The activity of the purified enzyme is independent of divalent cations, but NH4+, K+, Li+, and Na+ show about 20% stimulation of enzyme activity. The enzyme has two absorption maxima (280 and 420 mµ). The removal of pyridoxal phosphate from the enzyme is accompanied by the loss of the 420 mµ absorption. The enzyme contains 0.99 mole of pyridoxal phosphate per 100,000 g of protein. The minimum molecular weight computed on the basis of pyridoxal phosphate content and sedimentation coefficient is 200,000.