The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[2H]indole

The bacterial tryptophan synthase α2β2 complex catalyzes the final reactions in the biosynthesis of l-tryptophan. Indole is produced at the active site of the α-subunit and is transferred through a 25–30 Å tunnel to the β-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner [Eur. J. Biochem. 129 (1983) 571] proposed a two-step nucleophilic addition–tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[2H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4–2.0 in the reaction of 3-[2H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an α-subunit ligand, disodium α-glycerophosphate (Na2GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[2H]indole with tryptophan synthase preincubated with l-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the l-Trp external aldimine at 423 nm. The addition of Na2GP dramatically slows the rate of reaction of indole with the α-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[2H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na2GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na+ or Na2GP. These results support the previously proposed mechanism for the β-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.