Allosteric Regulation of Substrate Channeling in Tryptophan Synthase:  Modulation of the l-Serine Reaction in Stage I of the β-Reaction by α-Site Ligands^,

Abstract

In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].

Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

“…Indeed, in structures with IGP bound to the a site, the side chain of aE49 is either folded away in an inactive conformation, H bonded to the OH of aY173 (see for example ref. [5]), or is H bonded via one carboxylate oxygen to the hydroxyl of IGP, and thus appears to be ionized. These structures correspond to inactive conformations.…”

“…This finding strongly corroborates the proposal that bE109 facilitates nucleophilic attack of indole on EA C H T U N G T R E N N U N G (A-A) by stabilizing charge development on the indole ring as the CÀC bond is formed to give E(Q 2 ) as shown in Figure 2 B. [7] Comparison of open, inactive b-subunit structures of EA C H T U N G T R E N N U N G (Aex 1 ) complexes [5] with the two closed b-subunit conformations available for the wild-type enzyme, the (GP)EA C H T U N G T R E N N U N G (A-A) complex, [5] and the (indoline-G3P)E(Q indoline ) complex presented herein, reveals the structural switch from the open to the closed conformation of the b subunit (Figure 2 C). The formation of EA C H T U N G T R E N N U N G (A-A) entails the loss of the hydroxyl group of EA C H T U N G T R E N N U N G (Aex 1 ).…”

“…Probably, this causes the conformational switch in the following way: removal of the EA C H T U N G T R E N N U N G (Aex 1 ) hydroxyl causes the loss of its H bond to the bD305 carboxylate; this results in a repositioning of bD305 to form a salt bridge with bR141, and causes formation of a Hbond network between bS299-bS297-bD305-b141-bD138-bL166 (Figure 2 C). [5,8,9] This then leads to a large motion of the COMM domain. [8,9] Moreover, it pulls bL166 into the active site; this completes the binding pocket for the indole, which then, in stage II of the b reaction, is channeled to the b site and reacts with EA C H T U N G T R E N N U N G (A-A) to give the E(Q 2 ) intermediate (Scheme 1 A), possibly assisted by bE109 as explained before.…”

“…The COMM domain changes position dramatically as the reactions proceed, and the switch between low-and high-activity states is accompanied by transitions between open and closed conformations of the a and b subunits. [1,5] To study the mechanisms of the a and b reactions and their allosteric regulation, we determined the 2.1 resolution crystal structure (Table S1) Figure S2). …”

“…The indoline C-3 corresponds to the indole nitrogen in IGP (Figures 1 C, E), and thus aD60 is positioned to stabilize the positive charge that develops on the indole nitrogen during CÀC bond cleavage in IGP (Figure 1 D). [5] with the closed, active (indoline-G3P)E(Q indoline ) complex (gray). The PLP rings are superimposed to show movements relative to the coenzyme.…”