Kinetic studies of tryptophan synthetase. Interaction of substrates with the B subunit

Faeder, Edward J.; Hammes, Gordon G.

doi:10.1021/bi00823a003

Cited by 117 publications

(76 citation statements)

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“…The same prediction can now be made for reaction (2) catalyzed by the a-subunit. The analysis of the steady-state kinetics of reaction (3) catalyzed by the B,-subunit favoured an ordered-addition nonequilibrium mechanism, because indole was shown to have only a low affinity for the enzyme [17]. This has been confirmed here.…”

Section: Discussionsupporting

confidence: 72%

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The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

Weischet¹,

Kirschner²

1976

European Journal of Biochemistry

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The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual a and P,-subunits and to the a2 8,-cornplex of tryptophan synthase was studied by equilibrium dialysis. The use of ['4C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the a-subunit.The binding of indole to the active site of the a-subunit is weak (Kd = 18 mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the a-subunit. The B,-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The a,P,-cornplex retains one or two sites per ap-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component a and 8-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first. +L-tryptophan + H20. (reaction 3 )The a-subunit is capable of catalyzing reaction (2) and the P,-subunit reaction (3) albeit only 1 -2 % as efficiently as the a,P,-complex. Reactions (2) (3) can formally be considered to be partial reactions of the physiologically relevant reaction (I), which can be catalyzed only by the native a,P,-complex [I]. This hypothesis implies that indole is a free intermediate. However, no indole could be detected in various trapping experiments designed to prove its transient accumulation in the course of the reaction (1) [ 2 , 3 ] . These findings have been interpreted in terms of a channelling effect, whereby the particular organization of a hypothetical composite active site [4] precludes the equilibration of indole with the bulk solvent. However, this is not a unique explanation and more direct evidence pertaining to the relationship between the partial reactions (2) and ( 3 ) and the overall reaction (1) appears desirable. In particular, it is pertinent to ask whether the indole subsites in the active sites of the a and the P2-subunits are retained and/or modified in the a,~,-complex. Such studies would also provide ancillary evidence for interpreting steady-state kinetic data.

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Section: Discussionsupporting

confidence: 72%

“…However, both fluorescence [8] and absorbance [17] measurements have failed to detect indole binding to the enzyme in the absence of L-serine. Equilibrium dialysis studies with ['4C]indole expectedly showed that the affinity of the &subunit for indole is weak (Fig.…”

Section: The Binding Of Indole To the Andsubunit Of Tryptophan Synthasementioning

confidence: 99%

The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

Weischet¹,

Kirschner²

1976

European Journal of Biochemistry

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“…The protein concentration was determined spectrophotometrically (e 0.1 %, 1 cm, 280 nm were 2.37 for unfolded lysozyme and 0.58 for β2) The enzymatic activity of β2 was determined according to Faeder and Hammes [17]. Where needed, the inhibitory effect of the residual NDSB present in the assay mixture (determined by measuring the activity of native β2 in the presence of NDSB added at the desired concentration to the assay mixture) was taken into account to determine the renaturation yields.…”

Section: Methodsmentioning

confidence: 99%

Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

Vuillard

Rabilloud

Goldberg

1998

European Journal of Biochemistry

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Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21Ϫ27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of . Hen lysozyme and the β2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256 ; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB256 ; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.Keywords : sulfobetaine; protein folding; lysozyme; tryptophan synthase.Understanding the molecular mechanisms of protein folding is one of the most challenging problems of modern molecular and cell biology. Besides its academic interest it is also a major issue in biotechnology, since overexpressed proteins are often obtained as insoluble aggregates that must be submitted to complex procedures aimed at generating their biologically active conformation. These procedures, as well as basic investigations on the folding mechanisms, are frequently severely hampered by the tendency of the denatured protein to aggregate when transferred into the renaturation buffer. It has been proposed [1Ϫ3] that during their folding, proteins undergo a kinetic partitioning between two alternative pathways, one leading to the native conformation, the other to aggregates. In this model, it is also assumed that the energy barrier between these two final states is high enough to render their interconversion practically impossible. Based on this conclusion, a variety of approaches has been used to favor renaturation over aggregation [4]. Most methods aim at minimizing aggregation, rather than at helping productive folding. For instance, one can, lower the protein concentration to reduce the rate of multimolecular reactions involved in aggreCorrespondence to M. E. Goldberg, Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, FranceAb...

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“…The conversion of indole and serine to tryptophan (B-reaction; Fig. 1) catalyzed by tmTrpB1 or tmTrpB2 was measured at 80°C by absorption spectroscopy and analyzed using ⌬⑀ 290 (Trp Ϫ indole) ϭ 1.89 mM Ϫ1 cm Ϫ1 (40). Initial velocities were measured as a function of the concentration of either indole or serine, with the other substrate being present at saturating concentrations.…”

Section: Thermotoga Maritima Tryptophan Synthasementioning

confidence: 99%

A Novel Tryptophan Synthase β-Subunit from the HyperthermophileThermotoga maritima

Hettwer

Sterner

2002

Journal of Biological Chemistry

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Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an ␣␤␤␣ complex in mesophilic microorganisms. The ␣-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated ␤-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in the trp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA, trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar ␣␤␤␣ complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a ␤ 2 -homodimer that has a high catalytic efficiency k cat /K m indole because of a very low K m indole but does not bind to TrpA. These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles.

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Kinetic studies of tryptophan synthetase. Interaction of substrates with the B subunit

Cited by 117 publications

References 14 publications

The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

A Novel Tryptophan Synthase β-Subunit from the HyperthermophileThermotoga maritima

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Kinetic studies of tryptophan synthetase. Interaction of substrates with the B subunit

Cited by 117 publications

References 14 publications

The Binding of Indole to the α‐Subunit and β2‐Subunit and to the α2β2‐Complex of Tryptophan Synthase from Escherichia coli

The Binding of Indole to the α‐Subunit and β2‐Subunit and to the α2β2‐Complex of Tryptophan Synthase from Escherichia coli

Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

A Novel Tryptophan Synthase β-Subunit from the HyperthermophileThermotoga maritima

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The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli