Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase ( R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718–4723 ). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a K D of 0.03 m m, which is comparable to the K 1 for inhibition of pyruvate ion formation (0.01 m m) and the K m for l-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 m m indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, k cat k inact , is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 m m indole and trifluoroalanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300–310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthasetrifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indolelyase occurs via a catalytically competent species, probably the β,β-difluoro-α-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.