Subunit structure of aspartate transcarbamylase from Escherichia coli.

1. The molecular weights of the catalytic and regulatory polypeptide chains of aspartate transcarbamylase were determined to be 33,500 and 17,000, respectively. The results of three empirical methods are in good agreement with those obtained by sedimentation equilibrium in guanidine-hydrochloride or derived from the intrinsic viscosity and the sedimentation coefficient measured in the same solvent. For the regulatory chain, all values obtained are in the range of 17,000, in agreement with the amino acid sequence (WEBER, K., Nafure, 218, 1116 (1968)). 2. The molecular weights of the native enzyme and the catalytic subunit were redetermined. The two proteins have masses of 300,000 and 100,000 daltons and show no association-dissociation equilibrium. From the molecular weights of the catalytic subunit and its polypeptide chain, we conclude that this subunit is a trimer. 3. Three independent techniques indicate that the catalytic and the regulatory polypeptide chains are present in equal numbers. This result, in combination with the molecular weights of these chains, indicates that the enzyme molecule contains six catalytic and six regulatory polypeptide chains which are held together solely by noncovalent bonds. 4. Renaturation of aspartate transcarbamylase and the catalytic subunit was achieved from guanidine hydrochloride or dodecyl sulfate solutions. The reactivated native enzyme and catalytic subunit are indistinguishable from their untreated counterparts. A subunit identical in every respect with catalytic subunit isolated after p-hydroxymercuribenzoate dissociation can also be obtained by heat treatment of the native enzyme. From these results it can be concluded that the catalytic subunit is preserved as a trimer in native aspartate transcarbamylase. A three dimensional arrangement of the enzyme with a Da symmetry is proposed. 5. The determination of the number of binding sites for the substrate analogue succinate in the catalytic subunit yields a value in agreement with the proposed model. Further support for a hexameric structure comes from the fact tnat asp&ate tiansciuaamyhaa ~.ati&rs & t&++U