Proton transfer and carbon-carbon bond cleavage in the elimination of indole catalyzed by Escherichia coli tryptophan indole-lyase

Tryptophan indole-lyase from Escherichia coli catalyzes the reversible clea vage of L-tryptophan to indole and ammonium pyruvate. This reaction is mech anistically interesting since it involves the elimination of an aromatic ca rbon leaving group. We have been studying the mechanism of tryptophan indol e-lyase using rapid-scanning stopped-flow spectrophotometry. Recently, we d emonstrated that the rate constant fur alpha-aminoacrylate intermediate for mation 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 confi rmed 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 +/- 0.6 7) on the elimination of indole. Tn addition, there is a significant solven t isotope effect (Dk = 1.79 +/- 0.11) on indole elimination in D2O. This so lvent isotope effect combines with the effect uf alpha-deuterium, since eli mination of alpha-H-2-L-tryptophan in DzO exhibits Dk = 4.30 +/- 0.16, In a ddition, the rate constant for indole elimination shows a linear Eyring plo t between 5 and 35 degrees C. In the direction of tryptophan synthesis, the reaction of the alpha-aminoacrylate intermediate with indole to form a qui nonoid 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 sug gest that the proton transfer and carbon-carbon bond cleavage in tile elimi nation reaction: are Yes nearly simultaneous and that the indolenine struct ure is a transient intermediate which occupies a very shallow well an the r eaction coordinate, or a transition state, in the reaction of Trpase.