Stereochemistry and Mechanism of Reactions Catalyzed by Tryptophanase from Escherichia coZi*

Several p replacement and cu,p elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)and (2S,3S)-[3-3H]serine in the presence of indole, deamination of these serines in DzO to pyruvate and ammonia, and cleavage of (2S,3R)and (2S,3S)-[3-3H]tryptophan in DzO to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,SH]pyruvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer ‘of the (Y proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactorsubstrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff’s base. Degradation of the resulting pyridoxylalanine to (2S)-[2-3H]alanine and (4’S)-[4’-3H]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4’ of the complex and that the ketimine double bond is trans.