Mechanisms of Monovalent Cation Action in Enzyme Catalysis: The Tryptophan Synthase α-, β-, and αβ-Reactions

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Abstract
The α-subunit of the tryptophan synthase bienzyme complex catalyzes the formation of indole from the cleavage of 3-indolyl-d-glyceraldehyde 3‘-phosphate, while the β-subunit utilizes l-serine and the indole produced at the α-site to form tryptophan. The replacement reaction catalyzed by the β-subunit requires pyridoxal 5‘-phosphate (PLP) as a cofactor. The β-reaction occurs in two stages: in stage I, the first substrate, l-Ser, reacts with the enzyme-bound PLP cofactor to form an equilibrating mixture of the l-Ser Schiff base, E(Aex1), and the α-aminoacrylate Schiff base intermediate, E(A-A); in stage II, this intermediate reacts with the second substrate, indole, to form tryptophan. Monovalent cations (MVCs) are effectors of these processes [Woehl, E., and Dunn, M. F. (1995) Biochemistry 34, 9466−9476]. Herein, detailed kinetic dissections of stage II are described in the absence and in the presence of MVCs. The analyses presented complement the results of the preceding paper [Woehl, E., and Dunn, M. F. (1999) Biochemistry 38, XXXX−XXXX], which examines stage I, and confirm that the chemical and conformational processes in stage I establish the presence of two slowly interconverting conformations of E(A-A) that exhibit different reactivities in stage II. The pattern of kinetic isotope effects on the overall activity of the β-reaction shows an MVC-mediated change in rate-limiting steps. In the absence of MVCs, the reaction of E(A-A) with indole becomes the rate-limiting step. In the presence of Na+ or K+, the conversion of E(Aex1) to E(A-A) is rate limiting, whereas some third process not subject to an isotope effect becomes rate determining for the NH4+-activated enzyme. The combined results from the preceding paper and from this study define the MVC effects, both for the reaction catalyzed by the β-subunit and for the allosteric communication between the α- and β-sites. Partial reaction-coordinate free energy diagrams and simulation studies of MVC effects on the proposed mechanism of the β-reaction are presented.