ALLOSTERIC INTERACTIONS IN ASPARTATE TRANSCARBAMYLASE .I. BINDING OF SPECIFIC LIGANDS TO NATIVE ENZYME AND ITS ISOLATED SUBUNITS

The regulatory enzyme, aspartate transcarbamylase (ATCase), which in earlier studies was shown to be composed of two catalytic and four regulatory subunits, was examined with regard to its stereospecific interactions with ligands. Equilibrium dialysis experiments reveal four specific binding sites for succinate (an analog of the substrate, aspartate) and four specific sites for the feedback inhibitor, cytidine triphosphate (CTP) or its analog, 5-bromocytidine triphosphate (BrCTP) per molecule of enzyme. On the basis of these results and evidence from dissociation studies, ATCase is viewed as an isologous tetramer, the protomers of which each contain one regulatory subunit (mol wt 2.7 × 104) and one-half of a catalytic subunit (mol wt 5.0 × 104). It was found that the isolated regulatory subunit possessed one binding site for CTP and the isolated catalytic subunit (mol wt 1 X 105) possessed two sites for succinate. Whereas the binding of ligands to the isolated subunits is normal, unusual effects are observed for the binding of these same ligands to the native enzyme. ATCase exhibits cooperative effects for succinate binding as revealed by a sigmoidal saturation curve (with a Hill coefficient of 1.6) and antagonistic effects as revealed by a partial reduction of CTP binding by succinate. It is concluded that the binding of succinate and CTP occurs at topographically distinct sites derived exclusively from the folded polypeptide chains of the different subunits, and that the cooperative and antagonistic effects are therefore indirect, i.e., allosteric effects, which must be mediated by the protein itself. In contrast to these indirect effects, ATCase also exhibits a direct effect in which the inhibitor (CTP) and the activator (adenosine triphosphate, ATP) appear to compete for a single site on the regulatory subunit. © 1968, American Chemical Society. All rights reserved.