The mechanisms of hydrolysis of the 2-halotryptophans at low pH have now been fully elucidated and reveal the involvement of intramolecular proton transfer in the conversion of the stable indole to the labile indolenine tautomer. An enzyme carboxyl group should also promote indolenine formation, suggesting the indolenine to be the true substrate for certain tryptophan enzymes. The first conclusive support for this concept is found in the demonstration that 2,3-dihydro-L-tryptophan and oxindolyl-L-alanine, analogs of the indolenine tautomer of tryptophan (tetrahedral carbon at C-3), are potent competitive inhibitors of tryptophan synthase and tryptophanase. Furthermore, the two enzymes show opposing specificity for the C-3 diastereoisomers of 2,3-dihydro-L-tryptophan, suggesting that these enzymes catalyze their reactions via enantiomeric indolenine intermediates. Fluorine-19 nuclear magnetic resonance and differential absorption spectroscopy have been used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, 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, tryptophan, and (3S)-2,3dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. These isomerization reactions are much slower than the Beta-replacement and Beta-elimination reactions catalyzed by tryptophan synthase. Since pyridoxal phosphate itself slowly catalyzes many reactions of amino acids in model systems, 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. Our results further define the stereochemistry of the substrate binding site of tryptophan synthase.
