We have examined the pre-steady-state kinetics of the reaction of Escherichia coli tryptophan indole-lyase with L-tryptophan, 7-aza-DL-tryptophan, and S-benzyl-L-cysteine. L-Tryptophan and 7-aza-DL-tryptophan exhibit three relaxations when the reactions are monitored at 506 nm. With L-tryptophan,-deuteriation results in an estimated isotope effect of 3.6 on the first phase, while 12H20 produces apparent isotope effects of 2.5 and 2.7 on the second and third phases, respectively. On the basis of the substrate and solvent isotope effects and the effects of aza substitution, we assign these three processes to (1) deprotonation of the a-carbon,(2) an enzyme conformational change, and (3) indole tautomerization. In contrast, S-benzyl-L-cysteine exhibits only one catalytically competent relaxation, monitored at 512 nm. The intrinsic isotope effect for the reaction of a-[2H]-S-benzyl-L-cysteine is estimated tobe 7.9.-Proton abstraction is 10-100-fold faster than catalytic turnover in these reactions; thus, tautomerizationof the indole ringof L-tryptophan may be partially rate-determining.