Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) from one enzyme active site to the next without allowing free diffusion of the metabolite. Channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Although there are numerous examples of sequential enzyme pairs in glycolysis and other biosynthetic pathways that are thought to exhibit channeling, this is as yet controversial. Tryptophan synthase is considered the best example for an enzyme displaying channeling behavior between subunits. Tryptophan synthase is an alpha 2 beta 2 tetrameric enzyme complex, which catalyzes the last two steps in the biosynthesis of L-tryptophan. The alpha subunit catalyzes the cleavage of indole-3-glycerol phosphate to indole and glyceraldehyde-3-phosphate; the beta subunit catalyzes the condensation of indole with serine to form tryptophan, in a reaction mediated by pyridoxal phosphate. The inability to trap free indole in the steady-state reaction and analysis of the kinetics of single turnover reactions have led to the postulate that indole may pass directly from the alpha to the beta site without diffusion through solution (Demoss, J. A. (1962) Biochim. Biophys. Acta 62, 279-293; Matchett, W. M. (1974) J. Biol. Chem. 249, 4041-4049). The crystal structure of tryptophan synthase from Salmonella typhimurium has provided additional support for substrate channeling by elucidating a 25-A hydrophobic tunnel connecting the two catalytic sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). The structure suggests that mutation of a residue lining the tunnel to a more bulky residue might impede or block the passage of indole during catalysis thus enabling detection of indole during a single enzyme turnover. A mutant of tryptophan synthase has been prepared in which one of the residues lining the tunnel, beta Cys-170, has been replaced with a bulkier tryptophan residue. Kinetic and structural analyses of the beta C170W mutant by rapid chemical quench methods and x-ray crystallographic analysis show both the transient formation of indole and the obstruction of the tunnel, thus providing direct evidence for the substrate channeling mechanism.