Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit

Abstract

A BSTR ACTThe lack of knowledge of the threedimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-Å crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analogbound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The f lexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.Integration of metabolic pathways and tight control of protein functions require allosteric regulation, whose hallmark is functional cooperativity. In their pioneering paper aimed at accounting for the properties of allosteric proteins, such as hemoglobin and regulatory enzymes, Monod, Wyman, and Changeux (1) postulated that these oligomeric proteins exist in two (or more) conformational states and that ligands control the equilibrium between a low-affinity or less active, taut (T state) form and a high-affinity or more active, relaxed (R state) quaternary structure. This two-state model has been shown to account for both the homotropic cooperativity and the heterotropic effects (inhibition and activation) exhibited by Escherichia coli aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase, carbamoyl phosphate:L-aspartate carbamoyltransferase; EC 2.1.3.2), which catalyzes the first committed step in pyrimidine biosynthesis (2-4). In addition, physical chemical measurements of the change in quaternary structure and catalytic activation promoted by substoichiometric amounts of the bisubstrate analog (5), N-(phosphonacetyl)-L-aspartate (PALA), were interpreted readily in terms of the model (2).In parallel with tests of the two-state model based on enzyme kinetics and solution studies of the ligand-promoted global conformational changes in ATCase, Lipscomb and his colleagues (6-9) conducted detailed crystallographic investigations of the wild-type enzyme and various mutant forms. The extensive data include high-resolution structures of unliganded ATCase (8), PDB ID 6atl, and the PALA-liganded enzyme (9), PDB ID 8atc. Large conformational differences between the free and PALA-bound forms of the holoenzyme were proposed to define the structural differences between the T and R states.Despite the extensive kn...

Natural selection of more designable folds: A mechanism for thermophilic adaptation

“…Strikingly, T. tengcongensis adapts by removing this enzyme altogether. Presumably, it accounts for this loss in the same way as does Arabidopsis thaliana: by utilizing a promiscuous enzyme, ornithine carbomyltransferase (43) (CD ϭ 4.81 (44), Z ϭ 4.1 similarity to mesophilic ODX), to fill the role of ODX. Thus, the thermophilic organisms above adapt at least in part by optimizing designability of the protein fold responsible for ornithine decarboxylation.…”

Still Looking for the Ivory Tower

“…In the meantime, Peter Beernink in my lab along with Jim Endrizzi in Tom Alber's group determined the structure of the C trimer (32). The results were surprising in that the active isolated trimers were more similar to the C trimers in the inactive T conformation of ATCase than the subunits in the active R state.…”

Structure of the catalytic trimer ofMethanococcus jannaschiiaspartate transcarbamoylase in an orthorhombic crystal form

“…Leu263 is in a disallowed region, as is often the case for active-site residues. This residue is also found in disallowed regions in the PALA-liganded and unliganded E. coli catalytic subunit (Endrizzi et al, 2000;Beernink et al, 1999) and holoenzyme (Jin et al, 1999;Stevens et al, 1990a,b) numbers of intermolecular contacts (complex 1, 229; complex 2, 138). The final map, contoured at 1.0, shows continuous density for most main-chain and side-chain atoms for both complexes with a few exceptions.…”

“…The variation in the planar angles between the crystal forms is large and may suggest high flexibility in solution and high reactivity even though the near-threefold symmetry in each trimer is maintained. The unliganded E. coli catalytic trimer shows variation of this angle within the same trimer and the threefold symmetry is not maintained (Beernink et al, 1999).…”