Conformational Changes in Aspartate Transcarbamylase

Replacement of Zn(II) by Cd(II) in aspartate transcarbamylase from Escherichia coli gives rise to an intense circular dichroism difference spectrum with a maximum ellipticity at 259 nm of 44,000 deg cm2 per dmole of Cd(II). This Cotton effect can be assigned to the Cd(II) chromophore and used as a sensitive spectral probe of metal site conformational changes. When circular dichroism difference spectra for carbamyl phosphate and succinate binding to Cd(II)-aspartate transcarbamylase are compared with the corresponding circular dichroism difference spectra for Zn(II)-aspartate transcarbamylase, perturbations of the Cd(II)-chromophore are apparent. Thus, conformational changes at metal ion binding sites in the regulatory subunits occur when substrate or substrate analogue molecules bind at active sites in the catalytic subunits. Conversely, replacement of Zn(II) by Cd(II) slightly alters the kinetic and binding parameters of native aspartate transcarbamylase. Thus, alteration of the metal ion binding site structure alters the conformation of the active site. These observations show that a coupling exists between conformational changes at metal ion sites in regulatory subunits and active sites in catalytic subunits. Consequently, conformational changes at metal ion binding sites may be involved in the allosteric mechanism of homotropic interactions. The binding of the feedback inhibitor, CTP, either to Cd(II)-regulatory subunit or to Cd(II)-aspartate transcarbamylase causes no change in the circular dichroism of the Cd(II)-chromophore. Thus, the metal ion does not seem to be directly involved either in the binding of CTP or in the mechanism of communicating heterotropic interactions. Since the metal ion sites are implicated in homotropic interactions, but not in heterotropic interactions, the allosteric mechanisms of these two effects must involve different sets of conformational changes. To assess the linkage between conformational changes at different loci in aspartate transcarbamylase, circular dichroism changes at different wave lengths were monitored during ligand titrations. For Cd(II)-aspartate transcarbamylase, the apparent carbamyl phosphate concentration required for half-maximal change is 0.32 mm at 280 nm (tyrosyl chromophore) but 0.10 mm at 256 nm (Cd(II)-chromophore). The succinate concentration required for half-maximal change is 0.20 mm at 256 nm (Cd(II)-chromophore) but 0.40 mm at 290 nm (tryptophanyl chromophore). These results demonstrate the existence of multiple conformational equilibria for the binding of these substrate ligands and suggest that allosteric interactions of aspartate transcarbamylase cannot adequately be described by a two-state model.