Allosteric and conformational transitions in the tryptophan synthase bienzyme complex, and the role of monovalent cation activation by probing the cation free state of the enzyme

The tryptophan synthase alpha2beta2 bienzyme complex is a wonderful model to study, allosteric regulation of catalysis, substrate channeling, and structure-function relationships. This system requires the presence of a Monovalent cation (MVC) for activation, and undergoes a conformational transition from and open conformation, to a closed conformation. Both the alpha- and beta-subunits undergo a conformational transition, and it has been shown that a transition of one subunit assists in lowering the energy of the transition of the other subunit. Furthermore, these conformational transitions are triggered by the formation of covalent chemical intermediates in the pathway of L-Trp synthesis. The cleavage of 3-indole-D-glycerol 3'-phosphate (IGP) to D-glyceraldehyde 3-phosphate (G3P) and indole (alpha-reaction) occurs only when both the alpha- and beta-subunits are in a closed conformation. The beta-subunit undergoes a conformational transition upon the elimination of the hydroxyl of L-Ser, forming the E(A-A) species, the species which reacts with indole generated in the alpha-reaction. This allosteric communication requires the binding of a MVC for proper function, and it is the objective of this dissertation to describe how the MVC binding alters this communication, and how cation size plays a role. The studies presented herein establish the following; (a) the binding of Cs+ appears to be stabilize the closed conformation of the beta-subunit while Na + appears to preferentially stabilize the open conformation, (b) the MVC-Free from of the enzyme is impaired mainly by displaying a decreased affinity towards beta-site nucleophiles, and greatly inhibiting stage II of the beta-reaction, (c) the reaction of L-Ser with the MVC-Free E(Ain), the is the existence of an inactive E(A-A) which is formed after the initial formation of the active branch of this species. This dissertation examines the structural effects exerted on the beta-active site via the MVC site, and sheds much light on the mechanism of MVC induced activation.