Mechanisms of Monovalent Cation Action in Enzyme Catalysis:  The First Stage of the Tryptophan Synthase β-Reaction

Abstract

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.

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…Actually, there is a third very slow phase, characterized by a small amplitude, that we neglected, considering it to be catalytically irrelevant, in agreement with other studies (19,22,30). The rate constants determined at pH 7.8 in the absence and presence of sodium or potassium ions are in good agreement with those observed previously (27,37,39). The amplitude of the first phase A 1 generally accounted for over 70% of the fluorescence change (Table I) and did not show any pH dependence.…”

“…The rate constants, determined at pH 7.8, are in good agreement with previous studies carried out at the same pH (27,37,39,57), showing that the decay of the external aldimine is slower in the presence of sodium ions with respect to a monovalent cation-free solution and is faster in the presence of potassium and cesium ions (Table I). On the basis of data collected at pH 7.8, it was proposed that in the absence of monovalent cations both the external aldimine and the ␣-aminoacrylate form are in the less active open conformation; in the presence of sodium ions the external aldimine is in a partially open conformation and the aminoacrylate is in a closed conformation, and in the presence of cesium ions the aminoacrylate is in the more active closed state (23,27,37,39). However, within a defined conformation cations are clearly able to modulate differently the reactivity of the ␤-subunit and its pH dependence.…”

“…It is interesting to note that the same pattern with similar pK a values holds when IAG, an allosteric effector bound to the ␣-subunit, is present both in the absence and presence of ions (Table IV). All rates are higher in the presence of IAG in keeping with the role of the allosteric effectors to stabilize the closed conformation in the presence of monovalent cations (27,37,39). On the basis of these data it seems more plausible that pK a1 is shifted to lower values due to specific effects of cesium ions on the ionizable residue either via direct or indirect interactions.…”

“…Surprisingly, no studies have addressed the pH dependence of the catalytic rates on the S. typhimurium enzyme, the form for which the three-dimensional structure of the internal aldimine (12,15,35), the external aldimine (35,36), and the ␣-aminoacrylate (12) has been determined. Recently, it was also found that monovalent cations and ␣-subunit ligands affect the distribution of intermediates as well as the catalytic properties of TS, triggering regulatory signals and stabilizing alternative open and closed conformations of the ␣-and ␤-subunits (23,27,(37)(38)(39)(40)(41). We have investigated the pH dependence of the catalytic rates in the absence and presence of monovalent cations and indole acetylglycine (IAG), a recently discovered ␣-subunit ligand (13,42).…”

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

“…Previous investigations of tryptophan synthase have demonstrated a primary kinetic isotope effect for the L-serine deaminase reaction of the ␤ 2 subunit at pH 7.8 (14), for the synthesis of L-tryptophan (20,41,42), and for the equilibrium distribution of intermediates in the reaction of L-serine (8) and in the reaction of L-serine and indole (26).…”