The Molecular Basis of Substrate Channeling

Abstract

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...

A Transport Metabolon

“…the glycolysis, citric acid, and urea cycles (52). Channeling allows for increased flux through the pathway as it limits the loss of intermediates by diffusion (53). In addition, a metabolon would allow the creation of specific pools of substrates.…”

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…Multienzyme supramolecular complexes, which self-assemble into spatially defined architectures, have been shown to be a promising approach to improve the efficiency of cascade reactions (19)(20)(21)(22). This spatial organization provides more stable enzyme structures, facilitates substrate channeling between active sites, and prevents the buildup of toxic intermediates (23)(24)(25).…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…The most elegant solution to handling such intermediates in nature is the construction of tunnels linking two active sites so that reactive or toxic intermediates are never released to the solvent (51). In the absence of a tunnel, a transient physical interaction that allows channeling of a product from the active site at which it is formed to the active site at which it is consumed is also an effective mechanism for sequestering problematic intermediates (52). Because these mechanisms are difficult to generate by protein engineering, a practical approach used by synthetic biologists is to overproduce the enzyme immediately downstream of the enzyme that produces the toxic intermediate.…”