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

Abstract

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.

α2β1 Integrin Is Not Recognized by Rhodocytin but Is the Specific, High Affinity Target of Rhodocetin, an RGD-independent Disintegrin and Potent Inhibitor of Cell Adhesion to Collagen

“…Nonspecific binding signals measured as ␣ 2 ␤ 1 binding to the blocking agent BSA were subtracted from the binding values for ␣ 2 ␤ 1 binding to native and denatured rhodocetin, respectively. The titration curves were linearized, and a K d value was determined according to the algorithm given by Heyn and Weischet (22).…”

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

“…inset Fig.2). Indolepropanol phosphate binds to the active site of the a-subunit [6,7]. Since the active site is likely to bind the substrate indole, albeit weakly, the simplest interpretation of the interaction between indolepropanol phosphate and indole binding to the sites of low affinity is direct competition.…”

Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

“…The inhibition towards indole is probably non-competitive, although competition cannot be strictly excluded. Moreover spectrophotometric titrations employing circular dichroism have shown that D-glyceraldehyde 3-phosphate binds to the free a subunit [7]. In contrast the enzyme has only weak affinity for indole at the active center [6].…”

“…In the case of indoleglycerol phosphate synthesis this hypothesis is supported by the following additional evidence. Circular dichroism studies have shown that aromatic amino acid sidechains of the a subunit are perturbed by bound glyceraldehyde 3-phosphate [7]. Moreover, the binding of indolepropanol phosphate to either the a subunit or to the a2p2 complex of tryptophan synthase appears to be accompanied by conformational changes 15 -7, 28,291.…”