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Substrate channeling is the process of direct transfer of an intermediate between the active sites of two enzymes that catalyze sequential reactions in a biosynthetic pathway (for reviews see Refs. 1 and 2). The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in a multienzyme complex.Substrate channeling has been proposed to decrease transit time of intermediates, prevent loss of intermediates by diffusion, protect labile intermediates from solvent, and forestall entrance of intermediates into competing metabolic pathways (2). Loss of an intermediate by diffusion may be especially important in the case of a neutral species, such as indole, which could escape from the cell by passive diffusion across cell membranes (2-5). Nevertheless, there has been considerable debate over whether channeling actually occurs and whether it is advantageous (5, 6).X-ray crystallographic studies on several enzyme complexes have revealed two molecular mechanisms for channeling. The discovery of an intramolecular tunnel in tryptophan synthase (7) provided the first molecular mechanism for channeling. For a long time this was a unique example, but recent structure determinations have revealed plausible evidence for tunneling of ammonia and carbamate in carbamoyl-phosphate synthase (CPS) 1 (8) and of ammonia in phosphoribosylpyrophosphate amidotransferase (GPATase) (9).The structure of dihydrofolate reductase-thymidylate synthase, however, showed no evidence for a tunnel between the two active sites (10). Neither are the two active sites adjacent to one another. Instead, there are positively charged residues along the surface between the active sites that form an electrostatic highway sufficient to channel the negatively charged dihydrofolate with high efficiency (10, 11).The focus of this minireview will be on the structural basis of channeling in the four enzyme structures cited above and on solution studies of these systems.2 Scheme I summarizes the reactions catalyzed by the four enzymes. A comparison of the structural and kinetic results reveals that these enzymes frequently exhibit allosteric interactions that synchronize the reactions to prevent the build-up of excess intermediate (12)(13)(14)(15)(16). TunnelingTryptophan Synthase-Tryptophan synthase catalyzes the last two reactions in the biosynthesis of L-tryptophan (Scheme I, A) (for reviews see . In bacteria, the two reactions are catalyzed by separate ␣ and ␤ subunits, which combine to form a stable multienzyme complex, (␣␤) 2 . In yeast and molds, the two reactions are catalyzed by separate ␣ and ␤ sites in a single bifunctional polypeptide chain, (␣-␤) 2 .3 Evidence that indole is not liberated as a free intermediate in the overall conversion of indole-3-glycerol phosphate and L-serine to L-tryptophan suggested that indole passes directly from the ␣ site to the ␤ site without release to the surrounding solvent (20 -23). The 2.8-Å resolution crystal structure of tryptophan synthase from Salmonella typhimurium showe...

Three-dimensional structures are reported for a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with either the substrate L-serine (betaK87T-Ser) or product L-tryptophan (betaK87T-Trp) at the active site of the beta-subunit, in which both amino acids form external aldimines with the coenzyme, pyridoxal phosphate. We also present structures with L-serine bound to the beta site and either alpha-glycerol 3-phosphate (betaK87T-Ser-GP) or indole-3-propanol phosphate (betaK87T-Ser-IPP) bound to the active site of the alpha-subunit. The results further identify the substrate and product binding sites in each subunit and provide insight into conformational changes that occur upon formation of these complexes. The two structures having ligands at the active sites of both alpha- and beta-subunits reveal an important new feature, the ordering of alpha-subunit loop 6 (residues 179-187). Closure of loop 6 isolates the active site of the alpha-subunit from solvent and results in interaction between alphaThr183 and the catalytic residue alphaAsp60. Other conformational differences between the wild type and these two mutant structures include a rigid-body rotation of the alpha-subunit of approximately 5 degrees relative to the beta-subunit and large movements of part of the beta-subunit (residues 93-189) toward the rest of the beta-subunit. Much smaller differences are observed in the betaK87T-Ser structure. Remarkably, binding of tryptophan to the beta active site results in conformational changes very similar to those observed in the betaK87T-Ser-GP and betaK87T-Ser-IPP structures, with exception of the disordered alpha-subunit loop 6. These large-scale changes, the closure of loop 6, and the movements of a small number of side chains in the alpha-beta interaction site provide a structural base for interpreting the allosteric properties of tryptophan synthase.

Monovalent cations activate the pyridoxal phosphate-dependent reactions of tryptophan synthase and affect intersubunit communication in the alpha2beta2 complex. We report refined crystal structures of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium in the presence of K+ at 2.0 angstrom and of Cs+ at 2.3 angstrom. Comparison of these structures with the recently refined structure in the presence of Na+ shows that each monovalent cation binds at approximately the same position about 8 angstrom from the phosphate of pyridoxal phosphate. Na+ and K+ are coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, and beta Gly-232 and to two or one water molecule, respectively. Cs+ is coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, beta Gly-232, beta Val-231, beta Gly-268 and beta Leu-304. A second binding site for Cs+ is located in the beta/beta interface on the 2-fold axis with four carbonyl oxygens in the coordination sphere. In addition to local changes in structure close to the cation binding site, a number of long-range changes are observed. The K+ and Cs+ structures differ from the Na+ structure with respect to the positions of beta Asp-305, beta Lys-167, and alpha Asp-56. One unexpected result of this investigation is the movement of the side chains of beta Phe-280 and beta Tyr-279 from a position partially blocking the tunnel in the Na+ structure to a position lining the surface of the tunnel in the K+ and Cs+ structures. The results provide a structural basis for understanding the effects of cations on activity and intersubunit communication.