Tryptophan Synthase

Miles, Edith Wilson; Ahmed, Salman; Hyde, C. Craig; Kayastha, Arvind M.; Yang, Xiang-Jiao; Ruvinov, Sergei B.; Lu, Zuliang

doi:10.1002/9783527615971.ch7

Cited by 6 publications

(5 citation statements)

References 108 publications

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“…As an ideal system for investigating the protein−protein interaction and ligand-mediated allosteric regulation, bacterial tryptophan synthase has been widely studied using genetic, biochemical, and kinetic methods ( − ). Tryptophan synthase catalyzes the last two steps in the biosynthesis of l -tryptophan.…”

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confidence: 99%

“…For these concepts, the structural changes in proteins are considered to be connected with the regulation of function. As an ideal system for investigating the protein-protein interaction and ligand-mediated allosteric regulation, bacterial tryptophan synthase has been widely studied using genetic, biochemical, and kinetic methods (9)(10)(11)(12)(13)(14)(15)(16). Tryptophan synthase catalyzes the last two steps in the biosynthesis of L-tryptophan.…”

mentioning

confidence: 99%

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Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,

et al. 2004

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When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.

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confidence: 99%

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Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,

et al. 2004

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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%

“…Various methods have been used to this aim, including X-ray crystallography (1-7), site-directed mutagenesis (8-12), 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).Crystallographic data provide information about the static characteristics, or equilibrium properties, in general. Yet, indirect information about the conformational flexibility of † This work was supported by NATO CRG 951240 and the Intramural NCI Program.…”

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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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“…1, we postulate a simple model (Scheme II) in which both forms of the enzyme (E-Ser and E-AA) show weak "binding" 4 of ethanol (ROH), up to N moles/mol to E-Ser and up to M moles/mol to E-AA; k 0 is the intrinsic equilibrium constant for the interconversion of ESer and E-AA and k 1 is the apparent average binding con-activity. We expect that solutions of ␤-mercaptoethanol may show similar nonideality and that nonideality may distort extrapolations of our results to the absence of cosolvent in both cases.…”

Section: Ethanol Alters the Distribution Of Enzyme-substratementioning

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Mechanism of Activation of the Tryptophan Synthase α2β2 Complex

Ahmed

McPhie

Miles

1996

Journal of Biological Chemistry

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To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase ␣ 2 ␤ 2 complex, we have determined the solvent effects of a co-substrate, ␤-mercaptoethanol, and of a model nonsubstrate, ethanol, on the catalytic and spectroscopic properties of the enzyme. Our results show that ethanol and ␤-mercaptoethanol both alter the equilibrium distribution of pyridoxal 5-phosphate intermediates formed in the reactions of L-serine at the ␤ site in the ␣ 2 ␤ 2 complex. Addition of increasing concentrations of ethanol increases the proportion of the external aldimine of L-serine and decreases the proportion of the external aldimine of aminoacrylate. Low concentrations of the co-substrate ␤-mercaptoethanol (K d ‫؍‬ ϳ13 mM) decrease the proportion of the external aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of ␤-mercaptoethanol decrease the concentration of the quinonoid intermediate and increase the proportion of the external aldimine of L-serine. Data analysis shows that ␤-mercaptoethanol and ethanol both interact or bind preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shift the equilibrium in favor of this conformer. We propose that a nonpolar region of the ␤ subunit, possibly the hydrophobic indole tunnel, becomes less exposed to solvent in the conformational transition that activates the ␣ 2 ␤ 2 complex.To fully understand the relationships between enzyme structure and function, it is important to determine how enzyme activity is regulated by protein-protein interaction and proteinligand interaction. Investigations employing spectroscopic techniques can furnish valuable insight into the regulation of catalytic intermediates and of conformational states. The chromophoric pyridoxal 5Ј-phosphate (PLP) 1 coenzyme provides an excellent spectroscopic probe for detecting catalytic intermediates and monitoring the effects of ligands on the equilibrium distribution of enzyme-substrate intermediates formed by PLPdependent enzymes. Here we utilize the PLP coenzyme of the ␤ subunit of tryptophan synthase to investigate the activation of catalysis by the tryptophan synthase ␣ 2 ␤ 2 complex.The bacterial tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.2.1.20) is a bifunctional enzyme that catalyzes the final two reactions in the biosynthesis of L-tryptophan, termed the ␣ reaction (Equation 1) and the ␤ reaction (Equation 2) (for reviews, see Refs. 1-6).␣ Reaction: indole 3-glycerol phosphate 3 indole ϩ D-glyceraldehyde 3-phosphate (Eq. 1)These two reactions are catalyzed by the ␣ and ␤ subunits 2 in the ␣ 2 ␤ 2 complex and, at a much lower rate, by the isolated ␣ subunit and ␤ 2 subunit, respectively. The crystal structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (7) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel provides a plausible structural route for tra...

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Tryptophan Synthase

Cited by 6 publications

References 108 publications

Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,

Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Mechanism of Activation of the Tryptophan Synthase α2β2 Complex

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Tryptophan Synthase

Cited by 6 publications

References 108 publications

Conformational Changes in the α-Subunit Coupled to Binding of the β2-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone,

Conformational Changes in the α-Subunit Coupled to Binding of the β2-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone,

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Mechanism of Activation of the Tryptophan Synthase α2β2 Complex

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Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,

Conformational Changes in the α-Subunit Coupled to Binding of the β₂-Subunit of Tryptophan Synthase from Escherichia coli: Crystal Structure of the Tryptophan Synthase α-Subunit Alone^,