Tryptophan indole-lyase from Escherichia coli catalyzes the reversible cleavage of L-tryptophan to indole and ammonium pyruvate. This reaction is mechanistically interesting since it involves the elimination of an aromatic carbon leaving group. We have been studying the mechanism of tryptophan indole-lyase using rapid-scanning stopped-flow spectrophotometry. Recently, we demonstrated that the rate constant fur alpha-aminoacrylate intermediate formation from alpha-H-2-L-tryptophan exhibits an isotope effect of 3.0 (Sloan, M. J.; Phillips, R. S. Biochemistry 1996, 35, 16165-16173). We have confirmed this previous result ((D)k = 2.99 +/- 0.30) and we have now found that beta,beta-di-H-2-L-tryptophan also exhibits a secondary isotope effect (Dk = 1.17 +/- 0.03) on the elimination reaction. Furthermore, alpha,beta,beta-tri-H-2-L-tryptophan exhibits a multiple isotope effect (Dk = 4.42 +/- 0.67) on the elimination of indole. Tn addition, there is a significant solvent isotope effect (Dk = 1.79 +/- 0.11) on indole elimination in D2O. This solvent isotope effect combines with the effect uf alpha-deuterium, since elimination of alpha-H-2-L-tryptophan in DzO exhibits Dk = 4.30 +/- 0.16, In addition, the rate constant for indole elimination shows a linear Eyring plot between 5 and 35 degrees C. In the direction of tryptophan synthesis, the reaction of the alpha-aminoacrylate intermediate with indole to form a quinonoid intermediate also exhibits a kinetic isotope effect for 3-H-2-indole, with Dk = 1.88 +/-0.19. In contrast to our expectations, the results suggest that the proton transfer and carbon-carbon bond cleavage in tile elimination reaction: are Yes nearly simultaneous and that the indolenine structure is a transient intermediate which occupies a very shallow well an the reaction coordinate, or a transition state, in the reaction of Trpase.