A 70‐amino acid zinc‐binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer

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-(phosphonacety1)-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, I3C 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 lo-' 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 Ki, 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.

A Cooperative Escherichia coli Aspartate Transcarbamoylase without Regulatory Subunits,

“…Yet, wild-type c 3 does not display homotropic cooperativity, even when associated with regulatory chains. The association of wild-type c 3 with full-length regulatory chains (c 3 r 6 ) or the zinc-binding domain of the regulatory chain (c 3 −(Zn) 3 ) − influences substrate affinity but does not restore homotropic cooperativity. These results suggest that both catalytic trimers are required for homotropic effects because homotropic interactions have been suggested to arise from substrate binding at sites on different c 3 subunits − .…”

“…A similar complex was formed between a catalytic trimer and the zinc-binding domain of the regulatory chain, which consists of 70 amino acids. This complex displayed properties similar to those of the R functional state of the holoenzyme, increased thermal stability compared to c 3 , hyperbolic dependence of initial velocity on aspartate concentration, and 50% increase in aspartate affinity, indicating that substantial changes occur at the active site of the catalytic trimer when it interacts with the zinccontaining polypeptide of the regulatory chain (21)(22)(23). The native enzyme exists in two forms with the low-affinity, lowactivity T state being due to constraints imposed by quaternary structure that must be overcome, upon ligand binding, by conformational changes to the R state.…”

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

“…Because these changes are concerted and each chain is involved in several protein-protein interactions (33-36), short-and long-range effects are difficult to distinguish. Although the subunits have been studied extensively in solution (16,20,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51), crystal structures of an isolated catalytic trimer of E. coli ATCase in the presence and absence of substrate analogs that would allow conformational changes restricted to the catalytic trimer to be identified have not previously been available.We report here the crystal structure of E. coli OTCase complexed with its bisubstrate analog N-(phosphonacetyl)-Lornithine (PALO) determined at 2.8-Å resolution. Active site residues that interact with the bound inhibitor are identified and the conformational change that accompanies substrate binding is described.…”