Stereochemistry and mechanism of reactions catalyzed by tryptophan synthetase and its beta2 subunit.

The synthesis of tryptophan from serine and indole or indoleglycerol phosphate catalyzed by native tryptophan synthetase or PZ protein is shown to proceed stereospecifically with retention of configuration at Cg. In the a$ elimination reaction of serine to give pyruvate and ammonia catalyzed by the fiz protein, the hydrogen from C, is transferred intramolecularly and without exchange with solvent protons to Cg, where it replaces the OH group with net retention of configuration. In a competing reaction, an abortive transamination in the presence of mercaptoethanol to give pyridoxamine and S-pyruvyl mercaptoethanol, the same proton is transferred to the extent of 70% to C-4’ of the cofactor when the reaction is carried out in DzO. Together with the finding of Dunathan and Voet (Dunathan, H. G. and Voet, J. G. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3888) that the cofactor is protonated from the si face, these data fully define the geometry of the substrate l coenzyme complex and the position of an essential base relative to it. Since no isotope effect was observed in the protonation of C, of tryptophan synthesized from indole and serine in 50% DzO, the base must be monoprotic. The known inability of the enzyme to degrade tryptophan by a$ elimination is not due to inability to remove H, of tryptophan; native enzyme and j3~ protein catalyze a hydrogen exchange of tryptophan at a rate of20% and 14%, respectively, of that of tryptophan synthesis.