The use of 6-(difluoromethyl)indole to study the activation of indole by tryptophan synthase

6-(Difluoromethyl)indole has been characterized and developed as a probe for the turnover of indole by the bifunctional enzyme, tryptophan synthase (alpha 2 beta 2). The neutral form of the indolyl species undergoes a slow and spontaneous hydrolysis to produce 6-formylindole with a rate constant (k1) of 0.0089 +/- 0.0001 min-1. The overall rate is independent of pH in the range of 3.5-10.5. Above pH 10.5, the observed rate increases are due to the high reactivity of the anionic form of the indole; deprotonation at N-1 accelerates hydrolysis by 10(4)-fold (k2, 97 +/- 2 min-1). The magnitude of this effect provides a technique for detecting the formation or stabilization of the anionic form of indole. 6-(Difluoromethyl)indole is recognized and processed by the beta subunit of tryptophan synthase. Selective inactivation of the beta subunit prevents enzymatic processing of 6-(difluoromethyl)indole. Chromatographic isolation and mass spectral analysis has identified 6-(difluoromethyl)tryptophan as the sole turnover product of the indolyl substrate. The lack of enzyme-promoted dehalogenation does not exclude the formation of an indole anion during turnover but rather the data suggest that rapid carbon-carbon bond formation (greater than 5300 min-1) prevents the accumulation of this anion.