Fluorescence studies of substrate and subunit interactions of the .beta.2 protein of Escherichia coli tryptophan synthetase

Goldberg, Michel; York, Sheldon S.; Stryer, Lubert

doi:10.1021/bi00850a045

Cited by 112 publications

(109 citation statements)

References 11 publications

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“…5A). Extrapolation of the straight line obtained in the double reciprocal plot yields the dissociation constant for L-serine: KdSerne = 2 X 10-2 M. Even though this value is significantly higher than that ( 10-3 M) determined, in a similar way, for native 132 (22), it can be concluded that nicked 132 has kept a binding site for L-serine.…”

Section: Resultsmentioning

confidence: 67%

“…4A), with two maxima at 335 and 417 nm characteristic of protein-bound pyridoxal-P, is similar to that of native holo-(32 (6). The nicked holo-(2 also exhibits fluorescence properties similar to those of the native enzyme (22): an emission maximum close to 500 nm and an excitation maximum at 412 nm (Fig. 4B).…”

Section: Resultsmentioning

confidence: 70%

“…Binding of L-Serine. The binding of L-serine to native holo-(32 causes a striking increase in the fluorescence intensity and a red shift of the excitation maximum of the bound cofactor (22). This fluorescence band, called the "aqua-band" (22), is also exhibited by the nicked enzyme.…”

Section: Resultsmentioning

confidence: 99%

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Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Högberg-Raibaud¹,

Goldberg²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Globular proteins often appear to consist of distinct compact "domains," and the assumption is frequently implicitly made that these domains correspond to intermediate structures in the folding process. If this assumption is correct, the polypeptide fragment that builds up a domain should be able to spontaneously fold into its native conformation even when isolated. In an attempt to isolate and study such a fragment, the #2 subunit of tryptophan synthetase [

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Section: Resultsmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 70%

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Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Högberg-Raibaud¹,

Goldberg²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Direct binding studies of indole to the a [5-71 and to the P,-subunits [8] have hitherto provided inconclusive evidence with regard to the number of sites per polypeptide chain. The availability of indolepropanol phosphate, a non-reactive analogue of the substrate indoleglycerol phosphate [6], allows competitive binding studies to be done for identifying a particular indole-binding site as being part of the active site of the a-subunit.…”

Section: (Reaction 3 )mentioning

confidence: 99%

“…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 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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Tryptophan Synthase: Structure, Function, and Subunit Interaction

Miles¹

1979

Advances in Enzymology - And Related Areas of Molecular Biology

145

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Fluorescence studies of substrate and subunit interactions of the .beta.2 protein of Escherichia coli tryptophan synthetase

Cited by 112 publications

References 11 publications

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

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

Tryptophan Synthase: Structure, Function, and Subunit Interaction

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Fluorescence studies of substrate and subunit interactions of the .beta.2 protein of Escherichia coli tryptophan synthetase

Cited by 112 publications

References 11 publications

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

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

Tryptophan Synthase: Structure, Function, and Subunit Interaction

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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