βD305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity

Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of βAsp 305, βArg 141, βLys 167, and αAsp 56 that appear to modulate the interconversion between open and closed conformations, βAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for βAsp 305 is to ensure the allosteric transmission that triggers the switching of αβ-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of βAsp 305 with Ala (βD305A) decreases the affinity of the β-site for the substrate L-Ser, destabilizes the enzyme-bound α-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the β-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. βD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the α-site substrate analogue, α-glycerol phosphate, the Na+ form of βD305A undergoes inactivation via reaction of nascent α-aminoacrylate with bound PLP. This work establishes important roles for βAsp 305 both in the conformational change between open and closed states that takes place at the β-site during the formation of the E(A-A) and in substrate binding and recognition.