Tryptophan Synthase

Miles, Edith Wilson

doi:10.1007/978-1-4899-1727-0_8

Cited by 54 publications

(9 citation statements)

References 152 publications

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“…Recently we have shown that both the α-subunits and the allosteric effector -α-glycerol 3-phosphate decrease the stereospecificity of tryptophan synthase [9]. -α-Glycerol 3-phosphate can be considered as either an analogue of -glyceraldehyde 3-phosphate or of indol-3-ylglycerol phosphate, which binds to the indol-3-ylglycerol phosphate binding site on the α-subunit [5,[10][11][12]. It has no effect on the catalytic properties of isolated β-subunits but order exchange rate of the slowly exchanged α-proton of alanine.…”

Section: Introductionmentioning

confidence: 99%

The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

Milne

Malthouse

1996

Biochemical Journal

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1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) c catalysed hydrogen-deuterium exchange of the alpha-protons of L- and D-alanine and -tryptophan. The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-alpha-glycerol 3-phosphate. In the presence of DL-alpha-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalyzed first-order exchange rates was in the order tryptophan > alanine > glycine. This increase in stereospecificity was largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton. A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton of D-alanine. Adding DL-alpha-glycerol 3-phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan. The alpha-subunits are shown to increase the exchange rates of the alpha-protons of L-alanine and L-tryptophan. We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its alpha-proton can be similar to or larger than that of its alpha-carboxylate group. Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed.

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Section: Introductionmentioning

confidence: 99%

The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

Milne

Malthouse

1996

Biochemical Journal

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“…The ␣ and ␤ activities are reciprocally modulated. Extensive functional and structural studies of the wild type enzyme and several mutants have unveiled some of the mechanisms underlying catalysis and allosteric regulation (3). In particular, the ␤ subunit exists either in a closed, catalytically active state, when the ␣-aminoacrylate is the most populated catalytic intermediate, or in an open, catalytically less active state, when the external aldimine is the predominant species (5)(6)(7)(8).…”

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confidence: 99%

Allosteric Communication of Tryptophan Synthase

Biase

Tramonti

Bettati

et al. 2001

Journal of Biological Chemistry

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The ␣ 2 ␤ 2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the ␤S178P mutant. Ser-178 is located at the end of helix 6 of the ␤ subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of ␤Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the ␣ subunit only when ␣ subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the ␤S178P mutation suggests that the hydrogen bond involving ␣Gly-181 is disrupted as a result of localized structural perturbations. The ratio of ␣ to ␤ subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight ␣؊␤ complex. Both the activity of the ␣ subunit and the inhibitory effect of the ␣ subunit ligands indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the ␤ subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate do not affect the ␤ activity. Accordingly, the distribution of L-serine intermediates at the ␤-site, dominated by the ␣-aminoacrylate, is only slightly influenced by ␣ subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the ␣-aminoacrylate tautomer absorbing at 450 nm. Crystals of the ␤S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the ␣ and ␤ activity in the ␣2␤2 complex with respect to the isolated subunits results from interactions that involve residues different from ␤Ser-178 and (ii) ␤Ser-178 is a critical residue in ligand-triggered signals between ␣ and ␤ active sites.Allosteric proteins are key elements of cellular regulation, because they allow for a fine adaptation of the living organisms to changes in metabolite concentration and physico-chemical conditions. Tryptophan synthase is an allosteric enzyme, composed of two ␣ and two ␤ subunits, catalyzing the last two steps in the biosynthesis of L-tryptophan (1-3) (Fig. 1). The ␣ active site cleaves indole-3-glycerol phosphate into indole and D-glyceraldehyde-3-phosphate. The ␤ active site contains a pyridoxal 5Ј-phosphate molecule bound to Lys-87 via a Schiff base and catalyzes the replacement of the ␤-hydroxyl of L-serine with indole. The latter compound is intramolecularly channeled from the ␣ site via a hydrophobic tunnel connecting the ␣ and ␤ active sites, as proposed on the basis of the first crystal structure of the enzyme (4). The ␣ and ␤ activities are reciprocally modulated. Extensive functi...

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“…[1][2][3][4][5]. The ␤ subunit 1 is the prototypic member of a family of pyridoxal phosphate (PLP) 2 -dependent enzymes that have distantly related sequences and that catalyze ␤-replacement and ␤-elimination reactions (6 -8).…”

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confidence: 99%

Structure and Function of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles²

1999

Journal of Biological Chemistry

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To probe the structural and functional roles of activesite residues in the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium, we have determined the effects of mutation of The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.2.1.20) is a useful system for investigating relationships between protein structure and function, allosteric communication between subunits in a multienzyme complex, and substrate channeling (for reviews, see Refs. 1-5). The ␤ subunit 1 is the prototypic member of a family of pyridoxal phosphate (PLP) 2 -dependent enzymes that have distantly related sequences and that catalyze ␤-replacement and ␤-elimination reactions (6 -8). The crystal structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (9) suggested the probable fold of other members of this family. Recent structures of biosynthetic threonine deaminase from Escherichia coli (10) and of O-acetylserine sulfhydrylase from S. typhimurium (11) confirm that these enzymes have the same fold as the tryptophan synthase ␤ subunit.

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Tryptophan Synthase

Cited by 54 publications

References 152 publications

The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

Allosteric Communication of Tryptophan Synthase

Structure and Function of the Tryptophan Synthase α2β2 Complex

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