Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan

Publisher Website
Google Scholar
Abstract
The reactions of the indole analogues indoline and aniline with the Escherichia coli tryptophan synthase .alpha.-aminoacrylase Schiff base intermediate have been characterized by UV-visible and 1H NMR absorption spectroscopy and compared with the interactions of indole and the potent inhibitor benzimidazole. Indole, via the enamine functionality of the pyrrole ring, reacts with the .alpha.-aminoacrylate intermediate, forming a transient quinonoid species with .lambda.max 476 nm as the new C-C bond is synthesized. Conversion of this quinonoid to L-tryptophan is the rate-limiting step in catalysis [Lane, A., and Kirschner, K. (1981) Eur. J. Biochem. 120, 379-398]. Both aniline and indoline undergo rapid N-C bond formation with the .alpha.-aminoacrylate to form quinonoid intermediates; benzimidazole binds rapidly and tightly to the .alpha.-aminoacrylate but does not undergo covalent bond formation. The indoline and aniline quinonoids (.lambda.max 464 and 466 nm, respectively) are formed via nucleophilic attack on the electrophilic C-.beta. of the .alpha.-aminoacrylate. The indoline quinonoid decays slowly, yielding a novel, new amino acid, dihydroisotryptophan. The aniline quinonoid is quasi-stable, and no new amino acid product was detected. We conclude that nucleophilic attack requires the precise alignment of bonding orbitals between nucleophile and the .alpha.-aminoacrylate intermediate. The constraints imposed by the geometry of the indole subsite force the aromatic rings of indoline, aniline, and benzimidazole to bind in the same plane as indole; thus nucleophilic attack occurs with the N-1 atoms of indoline and aniline. However, in the case of benzimidazole, this orientation places the reactive lone pair of electrons (the nonbonding pair on N-3) orthogonal to the direction necessary for nucleophilic attack. Since the .pi.-system of the five-membered ring is too resonance stabilized to allow reaction to occur analogous to that of the indole enamine, benzimidazole is prohibited from reacting via stereoelectronic constraints.