The tryptophan synthase bienzyme complex transfers indole between the .alpha.- and .beta.-sites via a 25-30 .ANG. long tunnel

Dunn, Michael F.; Aguilar, Valentin; Brzović, Peter S.; Drewe, William Frederick; Houben, Karl; Leja, Catherine A.; Roy, M.

doi:10.1021/bi00489a015

Cited by 185 publications

(312 citation statements)

References 58 publications

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“…Various methods have been used to this aim, including X-ray crystallography (1-7), site-directed mutagenesis (8)(9)(10)(11)(12), and kinetic analyses (10,11,(13)(14)(15)(16)(17). For knowledgeable, informed descriptions of previous work, see the reviews of Miles and collaborators (3,(18)(19)(20), and Dunn and coworkers (21).…”

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confidence: 99%

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Bahar

Jernigan

1999

Biochemistry

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Tryptophan synthase (TRPS), with linearly arrayed subunits R R, catalyzes the last two reactions in the biosynthesis of L-tryptophan. The two reactions take place in the respective R-and -subunits of the enzyme, and the intermediate product, indole, is transferred from the R-to the -site through a 25 Å long hydrophobic tunnel. The occurrence of a unique ligand-mediated long-range cooperativity for substrate channeling, and a quest to understand the mechanism of allosteric control and coordination in metabolic cycles, have motivated many experimental studies on the structure and catalytic activity of the TRPS R 2 2 complex and its mutants. The dynamics of these complexes are analyzed here using a simple but rigorous theoretical approach, the Gaussian network model. Both wild-type and mutant structures, in the unliganded and various liganded forms, are considered. The substrate binding site in the -subunit is found to be closely coupled to a group of hinge residues ( 77-89 and 376-379) near the -interface. These residues simultaneously control the anticorrelated motion of the two -subunits, and the opening or closing of the hydrophobic tunnel. The latter process is achieved by the large amplitude fluctuations of the so-called COMM domain in the same subunit. Intersubunit communications are strengthened in the presence of external aldimines bound to the -site. The motions of the COMM core residues are coordinated with those of the R-hinge residues 174-179 on the interfacial helix H6 at the entrance of the hydrophobic tunnel. And the motions of H6 are coupled, via helix H1 and RL6, to those of the loop RL2 that includes the R-subunit catalytically active residue Asp60. Overall, our analysis sheds light on the molecular machinery underlying subunit communication, and identifies the residues playing a key role in the cooperative transmission of conformational motions across the two reaction sites.The tryptophan synthase (TRPS) 1 R 2 2 complex is a bifunctional enzyme that catalyzes the last two reactions in the biosynthesis of L-tryptophan (L-Trp). The bacterial enzyme structure consists of two R-and two -subunits arranged in an extended R R order (Figure 1a,b). The Rand -subunits contain the respective sites for the R-and -reactions producing L-Trp: the cleavage of indole 3-glycerol phosphate (IGP) to release indole and glyceraldehyde 3-phosphate (G3P) (R-reaction) and the conversion of indole to L-Trp via a condensation reaction with L-serine ( -reaction). The latter is mediated by the cofactor pyridoxal 5′-phosphate (PLP), which forms with L-Ser a quasi-stable R-aminoacrylate intermediate, E(A-A), highly reactive toward indole. The R -reaction resulting from the combination of presents a typical example of long-range allosteric effect in that the two reactions, occurring at two topologically distinct and distant sites, are highly coordinated through a PLPdependent activation-deactivation mechanism, and the metabolite, indole, is transferred from the active site in the R-subunit to the other in the -subuni...

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Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Bahar

Jernigan

1999

Biochemistry

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“…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)(8)(9)(10).…”

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confidence: 99%

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

Mozzarelli¹,

Peracchi²,

Rovegno³

et al. 2000

Journal of Biological Chemistry

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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...

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“…2 The UV-visible spectra of wild-type Trp synthase with L-Trp present (Fig. 3A) showed little change with NaCl, but an increase in the content of the quinonoid intermediate, absorbing at 476 nm, was seen in the presence of disodium ␣-GP, consistent with a closed conformation (11).…”

Section: Uv-visible Spectra Of Wild-type and Mutant Trp Synthase Withmentioning

confidence: 94%

“…1B). The binding of ␣-site ligands, disodium ␣-GP or D-glyceraldehyde 3-phosphate, results in a closed conformation of the ␣-site, which is transmitted allosterically to the ␤-active site and shifts the equilibrium at the ␤-site to favor the closed conformation (11). In the presence of disodium ␣-GP, the NMR signal was about twice as intense (Fig.…”

Section: Uv-visible Spectra Of Wild-type and Mutant Trp Synthase Withmentioning

confidence: 98%

“…X-ray crystallographic investigations of Trp synthase from Salmonella typhimurium have revealed a 25-30-Å long interenzyme tunnel linking the ␣-and ␤-active sites, through which it has been proposed that indole travels to the ␤-active site directly from the ␣-active site after formation (10 -14). Moreover, intermediate complexes of the ␣-and ␤-reactions reciprocally regulate catalysis at the other site in an allosteric manner (11,(15)(16)(17). These allosteric interactions coordinate the forward progress of the ␣␤-reaction.…”

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Detection of Open and Closed Conformations of Tryptophan Synthase by 15N-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance of Bound 1-15N-L-Tryptophan

Osborne

Teng

Miles

et al. 2003

Journal of Biological Chemistry

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(1-4). The enzyme is an ␣ 2 ␤ 2 complex, consisting of two ␣-subunits bound to a central ␤ 2 -dimer. Each ␣-subunit independently catalyzes the reversible aldol cleavage of indole-3-glycerol phosphate to indole and D-glyceraldehyde 3-phosphate (␣-reaction, Reaction 1). The ␤-subunit catalyzes the conversion of indole and Lserine to L-Trp, with PLP as the cofactor (␤-reaction, Reaction 2). The overall reaction is the combination of the ␣-reaction and the ␤-reaction (␣␤-reaction, Reaction 3). Indole is not observed as a free intermediate in the ␣␤-reaction (5-9), suggesting that indole is not released from the ␣-subunit of Trp synthase into solution during turnover; hence, Reaction 3 is the physiological reaction of Trp synthase. X-ray crystallographic investigations of Trp synthase from Salmonella typhimurium have revealed a 25-30-Å long interenzyme tunnel linking the ␣-and ␤-active sites, through which it has been proposed that indole travels to the ␤-active site directly from the ␣-active site after formation (10 -14). Moreover, intermediate complexes of the ␣-and ␤-reactions reciprocally regulate catalysis at the other site in an allosteric manner (11,(15)(16)(17). These allosteric interactions coordinate the forward progress of the ␣␤-reaction.In the absence of substrate, PLP is bound to ␤Lys87 in the form of the internal aldimine (E A ) (Scheme 1) (18). The reaction of L-Ser at the ␤-site, promoted by the presence of indole-3-glycerol phosphate bound at the ␣-active site, forms an external aldimine (E EA-Ser ), which is converted to an ␣-aminoacrylate Schiff's base (E AA ) with elimination of water, by the action of ␤Lys87 as a general base (19). In return, formation of the ␣-aminoacrylate Schiff's base with PLP at the ␤-site activates the ␣-reaction (20). D-Glyceraldehyde 3-phosphate bound to the ␣-site keeps the ␣-site closed, blocking the release of indole into solution and facilitating transfer of indole from the ␣-and ␤-active sites through the interenzyme tunnel. Nucleophilic addition of indole to the ␣-aminoacrylate intermediate at the ␤-site then affords the quinonoid intermediate of L-Trp (E Q ). ␤Glu109 activates the addition of indole, a weak nucleophile, probably by acting as a general base or H-bond acceptor to the N-H of the reacting indole (21). At this point, ␤Lys87 acts as a general acid, providing the proton for the conversion of E Q to the L-Trp external aldimine (E EA-Trp ) (19). Release of D-glyceraldehyde 3-phosphate from the ␣-site results in an open conformation, which then allows release of the L-Trp product from the ␤-active site. Nucleophilic attack of ␤Lys87 on E EA-Trp

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The tryptophan synthase bienzyme complex transfers indole between the .alpha.- and .beta.-sites via a 25-30 .ANG. long tunnel

Cited by 185 publications

References 58 publications

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

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

Detection of Open and Closed Conformations of Tryptophan Synthase by 15N-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance of Bound 1-15N-L-Tryptophan

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