Mechanism of tryptophan indole-lyase: insights from pre-steady-state kinetics and substrate and solvent isotope effects

Abstract

The pre-steady-state kinetics of the reaction of Escherichia coli tryptophan indole-lyase have been examined 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, {alpha}-deuteriation results in an estimated isotope effect of 3.6 on the first phase, while {sup 2}H{sub 2}O 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, these three processes have been assigned to (1) deprotonation of the {alpha}-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 {alpha}-({sup 2}H)-S-benzyl-L-cysteine is estimated to be 7.9. {alpha}-Proton abstraction is 10 to 100-fold faster than catalytic turnover in these reactions; thus, tautomerization of the indole ring of L-tryptophan may be partially rate-determining. 27 references, 3 figures.