The tryptophan synthase bienzyme complex is activated and regulated by the allosteric action of monovalent cations (MVCs). The kinetic dissection of the first stage (stage I) in the beta-reaction of tryptophan synthase, the reaction of L-serine with pyridoxal 5'-phosphate at the beta-site to give the alpha-aminoacrylate Schiff base intermediate, E(A-A), is here examined in the absence and presence of MVCs. This analysis reveals which of the individual steps are greatly affected in stage I and how the ground states and transition states are affected by MVCs. Kinetic studies in combination with a detailed relaxation kinetic analysis to determine the specific rate constants for the conversion of the L-Ser external aldimine, E(Aex1), to E(A-A) show that the primary kinetic isotope effect for proton abstraction from Calpha of the E(Aex1) species changes from 4.0 +/- 0.4 in the absence of MVCs to a value of 5.9 +/- 0.5 in the presence of Na+, indicating that the nature of the transition state for this C-H scission is significantly perturbed by the MVC effect. The E(A-A) species was found to exist in two conformations with different activities, the ratio of which is affected by the presence of MVCs. It is shown that changes in the rate constants of stage I are important in establishing the ratio of active to inactive conformations of the E(A-A) species. Consequently, the MVC effect alters the relative energies of both the transition states and the ground states for selected steps in stage I of the pathway. Hence, interactions at the MVC site give rise to a fine-tuning of the covalent bonding interactions between active site residues and the reacting substrate during the conformational cycle of the bienzyme complex.