Effect of pH and Monovalent Cations on the Formation of Quinonoid Intermediates of the Tryptophan Synthase α2β2 Complex in Solution and in the Crystal

Abstract

Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the ␤-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the ␣-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and ␤-mercaptoethanol. In solution, both monovalent cations (Cs ؉ or Na ؉ ) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when ␤-mercaptoethanol is used. As indoline and ␤-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless ␣-subunit ligands are also present. ␣-Subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.The bacterial tryptophan synthase ␣ 2 ␤ 2 complex catalyzes the last two steps in the biosynthesis of L-tryptophan (1-4). Indole is formed from the cleavage of indole-3-glycerol phosphate in the ␣-active site and subsequently is channeled, via a hydrophobic tunnel, to the ␤-active site (5, 6) where it is combined with L-Ser to make L-Trp. The reaction at the ␤-active site depends on the cofactor pyridoxal 5Ј-phosphate and proceeds through the formation of several intermediates, which are characterized by distinct absorption properties (Scheme 1) (2, 7-10).The ␣-and ␤-subunit activities are allosterically regulated (2-4, 6, 11-20) via the selective stabilization of ␣-and ␤-subunit conformations consisting of an "open," catalytically inactive state, and a "closed" catalytically active state (11,12,21,22). The internal and the external aldimines in the ␤-active site are in open and partially open conformations, respectively, and do not send regulatory signals to the ␣-active site (4, 12-14). The ␣-aminoacrylate, E(A-A), exists both in an open and a closed conformation depending on the presence of monovalent cations, whereas the quinonoid, E(Q 3 ), is predominantly in the closed state (13). Only the closed state of the ␤-subunit appears to be competent in the transmission of allosteric signals and, therefore, in stabilizing the cl...

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

“…The placement of S318 (solution pK a of ≥15) near the pyridine nitrogen (solution pK a of ∼5) raises questions about the protonation state of the ring nitrogen and its ability to stabilize a quinonoid species, which is not observed by stopped-flow spectroscopy. In tryptophan synthase, a member of the β-replacement subfamily, which also has a serine residue proximal to the pyridine nitrogen, a quinonoid intermediate is observed but only under alkaline conditions (pH > 9) or in the presence of metals such as CsCl (29). The presence of serine contrasts with the presence of a carboxylate side chain in the transaminases, which enhances the "electron sink" character of the pyridine ring and favors formation of the quinonoid intermediate (30).…”

Allosteric Communication of Tryptophan Synthase

“…In the wild type enzyme the reaction of the ␣-aminoacrylate with indoline leads to the accumulation of a quinonoid intermediate absorbing at 466 nm (16,27,28). When the ␤S178P enzyme reacts with indoline, a quinonoid species is formed, and the K d of indoline is similar (data not shown) to that of the wild type (16).…”

“…A well characterized effect of the ␣Ϫ␤ allosteric regulation is the change of the equilibrium distribution of catalytic intermediates at the ␤ active site caused by ␣ subunit ligands (2,5,8,15,16,20). This distribution is also affected by pH, monovalent cations, and temperature (8,21).…”

“…Specifically, ␣ subunit ligands, low pH, cesium ions, and high temperature stabilize the ␣-aminoacrylate intermediate, whereas high pH, sodium ions, and low temperature stabilize the external aldimine. Moreover, high pH and monovalent cations favor the accumulation of a quinonoid intermediate (16). To further evaluate the effect of mutation on the ␣Ϫ␤ regulation, the influence of ␣ subunit ligands, monovalent cations, and pH on the equilibrium distribution of catalytic intermediates of the ␤ active site was investigated.…”

“…Replacement of the suspending medium was carried out by passing solutions through the cell. The cell was placed on the stage of a Zeiss MPM03 microspectrophotometer, equipped with a 10 ϫ Zeiss UV-visible ultrafluar objective (15,16). Polarized absorption spectra were collected between 315 and 550 nm with the electric vector of the linearly polarized light parallel to the extinction directions on the (210) flat face of monoclinic crystals.…”