Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.

Brzović, Peter S.; Sawa, Yoshihiro; Hyde, C. Craig; Miles, Edith Wilson; Dunn, Michael F.

doi:10.1016/s0021-9258(18)42377-0

Cited by 76 publications

(82 citation statements)

References 41 publications

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“…These are proposed to control the R-β hinge bending motions, and also modulate the R-reaction. The involvement of RL2, and in particular that of RAsp60 in RL2, in the R-reaction catalysis, and in allosteric communication, has now been well-established by several experiments (9,16,36). Likewise, loop RL6 is important for ligand binding and communication between the Rand β-subunits.…”

Section: Discussionmentioning

confidence: 90%

“…In view of the occurrence of a unique ligand-mediated long-range cooperativity for substrate channeling between two distant reaction sites, and the more general interest of gaining an understanding of the mechanism of allosteric control and coordination in metabolic cycles, the structure and catalytic activity of the bacterial TRPS R 2 β 2 complex (EC 4.2.1.20) and its mutants have been extensively studied. 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%

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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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Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Bahar

Jernigan

1999

Biochemistry

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“…However, the experiments are not performed at equilibrium conditions. Typically (e.g., see refs − , ), they are started in the absence of products, and measurements are finished before a significant amount of products is accumulated.…”

Section: Single-molecule Stochastic Modelmentioning

confidence: 99%

“…Tryptophan synthase has been extensively experimentally investigated, both in terms of its kinetics and conformational dynamics. − In 1958, it was determined that the intermediate indole produced by cleavage of IGP is not released into solution . Spectroscopic properties of the pyridoxal phosphate (PLP) cofactor at one catalytic site have allowed the study of the kinetics of tryptophan synthase on short time scales.…”

Section: Introductionmentioning

confidence: 99%

“…The use of equilibrium, rapid mixing stopped-flow techniques and pressure and temperature jump relaxation methods have led to the formulation of a detailed chemical reaction mechanism. − In 1990, rapid-scanning stopped-flow and single-wavelength stopped-flow experiments have indicated that binding of a ligand at one catalytic site affects the catalytic activity and conformational environment at the second catalytic center . This has led to the studies where the kinetic − and stuctural − aspects of allostery in this enzyme were explored. Today, due to a large volume of data, tryptophan synthase plays a key role in the understanding of allostery, channeling, and intramolecular synchronization.…”

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Stochastic Analysis of Channeling Enzyme Tryptophan Synthase

2016

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The channeling enzyme tryptophan synthase provides a paradigmatic example of a chemical nanomachine. It possesses two active centers and, as a single molecule, catalyzes 13 different reaction steps with a complex pattern of allosteric regulation and with an intermediate product channeled from one active center to another. Here, the first single-molecule stochastic model of the enzyme is proposed and analyzed. All its transition rate constants were deduced from the experimental data available, and no fitting parameters were thus employed. Numerical simulations reveal strong correlations in the states of the active centers and the emergent synchronization of intramolecular processes in tryptophan synthase.

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Backbone entropy of loops as a measure of their flexibility: Application to a Ras protein simulated by molecular dynamics

Meirovitch

Hendrickson

1997

Proteins

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The flexibility of surface loops plays an important role in protein-protein and protein-peptide recognition; it is commonly studied by Molecular Dynamics or Monte Carlo stimulations. We propose to measure the relative backbone flexibility of loops by the difference in their backbone conformational entropies, which are calculated here with the local states (LS) method of Meirovitch. Thus, one can compare the entropies of loops of the same protein or, under certain simulation conditions, of different proteins. These loops should be equal in size but can differ in their sequence of amino acids residues. This methodology is applied successfully to three segments of 10 residues of a Ras protein simulated by the stochastic boundary molecular dynamics procedure. For the first time estimates of backbone entropy differences are obtained, and their correlation with B factors is pointed out; for example, the segments which consist of residues 60-65 and 112-117 have average B factors of 67 and 18 A2, respectively, and entropy difference T delta S = 5.4 +/- 0.1 kcal/mol at T = 300 K. In a large number of recent publications the entropy due to the fast motions (on the ps-ns time scale) of N-H and C-H vectors has been obtained from their order parameter, measured in nuclear magnetic resonance spin relaxation experiments. This enables one to estimate differences in the entropy of protein segments due to folding-unfolding transitions, for example. However, the vectors are assumed to be independent, and the effect of the neglected correlations is unknown; our method is expected to become an important tool for assessing this approximation. The present calculations, obtained with the LS method, suggest that the errors involved in experimental entropy differences might not be large; however, this should be verified in each case. Potential applications of entropy calculations to rational drug design are discussed.

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Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.

Cited by 76 publications

References 41 publications

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Cooperative Fluctuations and Subunit Communication in Tryptophan Synthase

Single-Molecule Stochastic Analysis of Channeling Enzyme Tryptophan Synthase

Backbone entropy of loops as a measure of their flexibility: Application to a Ras protein simulated by molecular dynamics

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