Serine and threonine desaminaes of Escherichia coli; activators for a cell-free enzyme.

Serine has been reported by many workers to be deaminated by a variety of bacterial cells and tissues, but this process was first studied in detail by Gale and Stephenson in 1938 (1). These workers followed the serine deaminase of Escherichia coli by measuring the release of ammonia by resting cell suspensions. The reaction proceeded anaerobically, thereby distinguishing it from the oxidative deaminases. Aging of cell suspensions caused a loss of deaminase activity, which could be prevented by the addition of reducing agents, such as glutathione or formate, or by adenylic acid. Chargaff and Sprinson (2, 3) studied serine and threonine deaminases, using toluene-treated suspensions of E. coli and found that pyruvate and a-ketobutyrate, respectively, accumulated as the products of anaerobic deamination. Neither the O-ethers of serine nor phosphoserine were deaminated anaerobically. On the basis of these findings, Chargaff and Sprinson suggested desaturation as the mechanism. Binkley (4) obtained cell-free extracts of serine deaminase from E. coli which were inactivated by dialysis. The activity was restored by the addition of zinc ions. Lichstein et al. (5, 6) in studying the metabolic r61e of biotin inactivated the serine and threonine deaminases of E. coli by aging cell suspensions in phosphate buffer at pH 4. The activity was restored by the addition of biotin or adenylic acid. With a cell-free preparation, only yeast extract caused partial reactivation. From this evidence it was suggested that a coenzyme form of biotin is present in yeast extract (7). In the present study, active serine and threonine deaminases have been obtained by growing E. coli in deep medium without carbohydrate. Vacuum-dried cells were prepared which contained most of the deaminase activity present in the living cells, and which differed from the living cells only in the requirement of adenylic acid for activation. The deaminases were freed from the cells by autolysis, and purified by ammonium sulfate precipitation and adsorption on calcium phosphate gel. The purified enzyme required both adenylic acid (AMP) and glutathione (GSH) for activity. The enzyme, as prepared, deaminated both serine and threonine. During serine deamination, simultaneous inactivation toward both substrates occurred.