We are studying the structure and the reaction mechanism of the bacterial tryptophan synthase Alpha2Beta2 complex. This multienzyme complex is the product of a well studied genetic system and is overproduced by some strains of bacteria. The chromophoric coenzyme, pyridoxal phosphate, serves as a reorter group of events at the active site of the Beta2 subunit and of changes in conformation of the Beta2 subunit. We are comparing the mechanism of tryptophan synthase with that of a closely related pyridoxal phosphate enzyme, tryptophanase. We find that these two enzymes share the same basic reaction mechanism but differ vastly in the rates of some of their individual reactions. They also differ in the steric course of formation of a reaction intermediate, the indolenine tautomer of L-tryptophan. Our studies of both enzymes have been facilitated by the use of a series of analogs of L- and D-tryptophan and of 5-fluoro-L- and D-tryptophan. Studies using 19F-NMR and difference spectroscopy have led to the discovery of two new isomerization reactions and of a cleavage reaction catalyzed by tryptophan synthase. These studies further define the stereochemistry and mechanism of tryptophan synthase. The tryptophan analogs are also useful in studies of the activity of crystals of the Alpha2Beta2 complex of tryptophan synthase from Salmonella typhimurium. We have demonstrated that microcrystals of the enzyme are active. Single crystals studied by microspectrophotometry show distinctive spectral changes in the presence of substrates and analogs. We plan to use these analogs in x-ray crystallographic studies after the structure of the native crystal is solved. Data collected at 2.8 Angstrom on the native crystal and on several metal derivatives is now being analyzed. We are engineering specific amino acid changes in tryptophan synthase by oligonucleotide-dependent site-specific mutagenesis. The first of these mutants in the Alpha subunit (Arg 179 to Leu) has been expressed, purified, crystallized, and is being characterized. Other mutants in which a cysteine is replaced with serine have been designed to help in the solution of the x-ray structure by removing specific mercury binding sites.
