Abstract
Interaction between a 70‐amino acid and zinc‐binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady‐state kinetics, heavy‐atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N‐(phosphonacetyl)‐L‐aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, ^13^C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain‐C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased “stickiness” of carbamoyl phosphate to the Zn domain‐C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain‐C trimer complex was about 10^–2^ that from C trimer. Additional evidence for a change in binding of carbamoyl phosphate as a result of interaction of zinc domain and C trimer was obtained from the pH profile for K~ia~ for carbamoyl phosphate. Whereas participation of only 1 protonated residue is implicated in binding of carbamoyl phosphate to C trimer, 2 residues must be ionized for binding to the Zn domain‐C trimer complex.