Circular dichroism and fluorescence studies on the binding of ligands to the alpha subunit of tryptophan synthase.
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.
D001667:Binding, Competitive; D002942:Circular Dichroism; D005453:Fluorescence; D005985:Glyceraldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000378:metabolism
D005994:Glycerophosphates; D006146:Guanidines; D007211:Indoles; D008024:Ligands; D005985:Glyceraldehyde; D014367:Tryptophan Synthase