Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy.

We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.