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 beta Asp 305, beta Arg 141, beta Lys 167, and alpha Asp 56 that appear to modulate the interconversion between open and closed conformations. beta Asp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for beta Asp 305 is to ensure the allosteric transmission that triggers the switching of alpha beta -dimeric units between open and closed conformations of low and high activity. This work shows that substitution of beta Asp 305 with Ala (beta D305A) decreases the affinity of the beta -site for the substrate L-Ser, destabilizes the enzyme-bound alpha -aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta -reaction. The altered specificity provides a biosynthetic route for new L-amino acids 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 alpha -site substrate analogue, alpha -glycerol phosphate, the Na+ form of beta D305A undergoes inactivation via reaction of nascent alpha -aminoacrylate with bound PLP. This work establishes important roles for beta Asp 305 both in the conformational change between open and closed states that takes place at the beta -site during the formation of the E(A-A) and in substrate binding and recognition.