Mechanisms of monovalent cation action in enzyme catalysis: the tryptophan synthase alpha-, beta-, and alpha beta-reactions

• Published in: Biochemistry (Easton) 1999-06, Vol.38 (22), p.7131-7141
• Main Authors: Woehl, E, Dunn, M F
• Format: Article
• Language: English
• Subjects: Catalysis; Cations, Monovalent - chemistry; Enzyme Activation; Enzyme Stability; Indoles - chemistry; Kinetics; Models, Chemical; Protons; Quaternary Ammonium Compounds - chemistry; Quinones - chemistry; Salmonella typhimurium - enzymology; Sodium Chloride - chemistry; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Time Factors; Titrimetry; Tryptophan Synthase - chemistry
• ISSN: 0006-2960
• DOI: 10.1021/bi982919p

The alpha-subunit of the tryptophan synthase bienzyme complex catalyzes the formation of indole from the cleavage of 3-indolyl-D-glyceraldehyde 3'-phosphate, while the beta-subunit utilizes L-serine and the indole produced at the alpha-site to form tryptophan. The replacement reaction catalyzed by the beta-subunit requires pyridoxal 5'-phosphate (PLP) as a cofactor. The beta-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 alpha-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 beta-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 beta-subunit and for the allosteric communication between the alpha- and beta-sites. Partial reaction-coordinate free energy diagrams and simulation studies of MVC effects on the proposed mechanism of the beta-reaction are presented.