The allosteric interactions that regulate substrate channeling and catalysis in the tryptophan synthase bienzyme complex from Salmonella typhimurium are triggered by covalent reactions at the β-site and binding of substrate/product to the α-site. The transmission of these allosteric signals between the α- and β-catalytic sites is modulated by an ensemble of weak bonding interactions consisting of salt bridges, hydrogen bonds, and van der Waals contacts that switch the subunits between open and closed conformations. Previous work has identified a scaffolding of salt-bridges extending between the α- and β-sites consisting of αAsp 56, βLys 167, and βAsp 305. This work investigates the involvement of yet another salt bridging interaction involving the βAsp 305-βArg 141 pair via comparison of the spectroscopic, catalytic, and allosteric properties of the βD305A and βR141A mutants with the behavior of the wild-type enzyme. These mutations were found to give bienzyme complexes with impaired allosteric communication. The βD305A mutant also exhibits altered β-site substrate reaction specificity, while the catalytic activity of the βR141A mutant exhibits impaired β-site catalytic activity. The >25-fold activation of the α-site by α-aminoacrylate Schiff base formation at the β-site found in the Na+ form of the wild-type enzyme is abolished in the Na+ forms of both mutants. Replacing Na+ by NH4+ or Cs+ restores the βD305A to a wild-type-like behavior, whereas only partial restoration is achieved with the βR141A mutant. These studies establish that the βD305-βR141 salt bridge plays a crucial role both in the formation of the closed conformation of the β-site and in the transmission of allosteric signals between the α- and β-sites that switch the α-site on and off.
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