THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX TRANSFERS INDOLE BETWEEN THE ALPHA-SITES AND BETA-SITES VIA A 25-30 A LONG TUNNEL

The bacterial tryptophan synthase bienzyme complexes (with subunit composition α2β2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the α-catalytic site to the β-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each α-β subunit pair are connected by a 25-30 Å long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the α-aminoacrylate Schiff base intermediate, E(A-A), bound to the β-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the α-site to the β-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D, L-α-glycerol 3-phosphate (GP) is bound to the α-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the β-site via the α-site and the tunnel, and this access is blocked by the binding of GP to the α-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with α2β2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the β-site of α2β2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the α- and β-sites. (3) The α-site and the interconnecting tunnel function as a highly preferred route for the transfer of indole and indole analogues such as benzimidazole, indoline, or aniline between solution and the β-catalytic site of E(A-A). (4) GP inhibits the access of these molecules to the β-site by blocking the tunnel opening at the α-site. (5) The entry of small nucleophiles into the β-site of E(A-A) from solution is not blocked by GP. (6) Either GP does not completely block the tunnel opening, or these small nucleophiles have an alternative route of access (perhaps through a leak in the tunnel wall or directly through the β-site).© 1990, American Chemical Society. All rights reserved.