Function of Pyridoxal Phosphate: Resolution and Purification of the Tryptophanase Enzyme of Escherichia Coli

Since Hopkins and Cole (1) demonstrated the formation of indole from tryptophan by Bacterium coli, numerous investigators have attempted to find the mechanism of this reaction (2-5). At least three mechanisms have been suggested. Woods (3) postulated an oxidat,ive degradation of tryptophan to yield indole, carbon dioside, water, and ammonia, 5 atoms of oxygen being used in the process. Baker and Happold (6) suggested a primary fission into indole and alanine, followed by the oxidation of alanine. I(rebs et al. (7) proposed that the mechanism involved a preliminary oxidation of the indole ring, followed by oxidation of the side chain to yield o-aminophenylacetaldehyde, which condensed to indole spontaneously. The data, however, did not substantiate this view and Krebs was led to state that, nhile Escherichia coli would form indole from o-amino-p-phenylethanol, via the analogous aldehyde, the mechanism of tryptophana.se action very probably did not involve this compound as an intermediate. Woods (3) and Baker and Happold (6) studied a series of possible oxidative intermediates between tryptophan and indole, and concluded that an unaltered alanine side chain was necessary for tryptophanase action. More recently, Dawson (8) found that mepacrine (atabrine) inhibits tryptophanase, and Dawes, Dawson, and Happold (9) have been able to recover alanine concurrently with indole formation in the presence of mepacrine. They have thus strengthened Baker and Happold’s postulate of primary fission to indole and alanine. In the present study, Escherichia coli cells with a very active tryptophanase system have been obtained by growing the culture with aeration. The cells have been vacuumor acetone-dried to yield cell preparations which contain most of the activity present in the living cells. The enzyme is stable in these preparations and may be obtained in a cell-free state by autolysis. The resolution and purification of the enzyme have been accomplished by precipitation of the cell-free extracts with ammonium sulfate and by calcium phosphate adsorption. Pyridoxal phosphate will reactivate the resolved enzyme, thereby adding tryptophanase to the group of vitamin Ba enzymes.