Propagation of conformational changes in Ni(II)-substituted aspartate transcarbamoylase: effect of active-site ligands on the regulatory chains.

Although the importance of ligand-promoted conformational changes in allosteric enzymes has been recognized, it often has been difficult to determine whether the effects of binding are propagated to remote positions in different chains. Efforts were made, therefore, to demonstrate that changes due to ligand binding to the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli are "communicated" to the regulatory chains. For these studies the endogenous zinc in the latter chains was replaced by nickel, which served as a discriminating spectral probe. The Ni(II)-enzyme was constructed by dissociating the native enzyme, separating the catalytic and regulatory subunits, removing Zn(II) from the latter, replacing it with Ni(II), and reconstituting the enzyme from native catalytic and Ni(II)-containing regulatory subunits. Ni(II) derivatives containing either six Ni(II) or five Ni(II) and one Zn(II) possess the allosteric properties of the native enzyme and exhibit absorption bands at 360 and 440 nm due to charge transfer transitions. Smaller bands were also observed at 665 and 720 nm from d-d transitions, which are consistent with tetrahedral geometry in the coordination sphere of nickel. Binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate to the catalytic subunit of Ni(II)-aspartate transcarbamoylase perturbed the Ni(II) chromophore, giving rise to two difference spectral bands (at 390 and 465 nm). Spectral titrations showed that the conformational changes at the metal-ion-binding sites were complete even though about one-third of the active sites were unoccupied. This propagation of conformational changes is in accord with other evidence indicating that the allosteric transition in aspartate transcarbamoylase is concerted.