Proton Transfer and Carbon−Carbon Bond Cleavage in the Elimination of Indole Catalyzed by Escherichia coli Tryptophan Indole-Lyase

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Abstract

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 for α-aminoacrylate intermediate formation from α-2H-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 (Dk = 2.99 ± 0.30) and we have now found that β,β-di-2H-l-tryptophan also exhibits a secondary isotope effect (Dk = 1.17 ± 0.03) on the elimination reaction. Furthermore, α,β,β-tri-2H-l-tryptophan exhibits a multiple isotope effect (Dk = 4.42 ± 0.67) on the elimination of indole. In 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 of α-deuterium, since elimination of α-2H-l-tryptophan in D2O exhibits Dk = 4.30 ± 0.16. In addition, the rate constant for indole elimination shows a linear Eyring plot between 5 and 35 °C. In the direction of tryptophan synthesis, the reaction of the α-aminoacrylate intermediate with indole to form a quinonoid intermediate also exhibits a kinetic isotope effect for 3-2H-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 the elimination reaction are very nearly simultaneous and that the indolenine structure is a transient intermediate which occupies a very shallow well on the reaction coordinate, or a transition state, in the reaction of Trpase.

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