Structural and Functional Roles of the Cysteine Residues in the α Subunit of the Escherichia coli Tryptophan Synthetase

Freedberg, W. B.; Hardman, John K.

doi:10.1016/s0021-9258(19)76992-0

Cited by 22 publications

(12 citation statements)

References 36 publications

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“…In the far-uv spectrum, no detectable change could be observed even at a concentration of 9 mM IND. This finding is consistent with one of Freedberg and Hardman (1971), who observed no change in the ORD spectrum of the enzyme in the presence of 10 mM IND in the wavelength range 350-600 nm.…”

Section: Resultssupporting

confidence: 93%

“…Possibly a very slow change in the protein conformation occurs. The binding of IND to the enzyme is known to be quite weak, with a dissociation constant of the order of several mM (Freedberg and Hardman, 1971). Because of the small induced CD effect, the large dissociation constant, and the large IND extinction coefficient, CD titration experiments could not be carried out, even with ultrathin cells (25 gm).…”

Section: Resultsmentioning

confidence: 99%

“…It can be used to study the binding via the indole group, and provides an additional method of determining binding constants. Conformational changes in the a subunit upon binding by IND and IGP have been postulated on the basis of biochemical and kinetic evidence (Freedberg and Hardman, 1971;Kirschner and Wiskocil, 1972;Yanofsky and Crawford, 1972). The possible existence of various conformations of the isolated a subunit also plays an important role in the assembly process of a and ß subunits into the multienzyme complex.…”

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confidence: 99%

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Circular dichroism and fluorescence studies on the binding of ligands to the α subunit of tryptophan synthase

Heyn¹,

Weischet²

1975

Biochemistry

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Binding to the alpha subunit of tryptophan synthase induces extrinsic Cotton effects in the substrates indole (IND), indoleglycerol phosphate (IGP), and D-glyceraldehyde-3-P (D-GAP) and in the inhibitor indolepropanol phosphate (IPP). These effects disappear when the enzyme is denatured in guanidinium chloride. The induced circular dichroism (CD) was used to determine the dissociation constant and the number of binding sites for IPP. The dissociation constant so determined is equal to 48 muM and is in good agreement with the value of 48 muM obtained by equilibrium dialysis. From the temperature dependence of the dissociation constant, a value of -2.8 kcal/mol for the binding enthalpy was obtained. The determination of dissociation constants by means of extrinsic Cotton effects is shown to be quite feasible. CD competition experiments with glycerol phosphate (GP) suggest that IPP binds bifunctionally to the enzyme: via its indole part and its phosphate group. Indolepropanol, which lacks the phosphate group, does not show an extrinsic Cotton effect. Since the induced CD is strongly dependent on the binding geometry, the close similarity between the induced spectra in IPP and IGP is additional evidence that IPP is a good substrate analog. Binding to the enzyme results in a blue shift of the IPP fluorescence emission maximum. The dissociation constant determined by fluorescence titration equals 46 muM and agrees well with the values determined by the other two methods. Previous biochemical and fast kinetic studies suggested the existence of multiple conformational states for the enzyme and of ligand-induced conformational changes. No evidence was found in the far-uv CD spectra for conformational changes upon binding of IND and D-GAP. For IPP a very small effect was observed.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Circular dichroism and fluorescence studies on the binding of ligands to the α subunit of tryptophan synthase

Heyn¹,

Weischet²

1975

Biochemistry

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“…A crude sonic extract of strain A2/F'A2 was used as a source of tryptophan synthetase ß subunit. 22 Protein concentration was determined by the method of Lowry, et al 23 Dialysis tubing was boiled in three changes of 5 X 10"3 M (ethylenedinitrilo)tetraacetic acid sodium salt, pH 7.0, before use in order to remove possible metal ion contaminants.…”

Section: Methodsmentioning

confidence: 99%

“…Chem., 242, 5442 (1967). of which appear to be located at or near the active site 21,22 jt has a molecular weight of 29,000, which corresponds roughly to the upper limit of molecular weight of enzymes which give proton nmr signals narrow enough to allow observation of a reasonable number of individual resonances.…”

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confidence: 99%

Studies of macromolecular structure by carbon-13 nuclear magnetic resonance. II. Specific labeling approach to the study of histidine residues in proteins

Browne¹,

Kenyon²,

Packer³

et al. 1973

J. Am. Chem. Soc.

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Conformational Adaptability in Enzymes

Citri¹

1973

Advances in Enzymology - And Related Areas of Molecular Biology

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Structural and Functional Roles of the Cysteine Residues in the α Subunit of the Escherichia coli Tryptophan Synthetase

Cited by 22 publications

References 36 publications

Circular dichroism and fluorescence studies on the binding of ligands to the α subunit of tryptophan synthase

Circular dichroism and fluorescence studies on the binding of ligands to the α subunit of tryptophan synthase

Studies of macromolecular structure by carbon-13 nuclear magnetic resonance. II. Specific labeling approach to the study of histidine residues in proteins

Conformational Adaptability in Enzymes

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