Explain the structural basis for cooperative substrate binding and allosteric control in ATCase.

Aspartate transcarbamoylase (ATCase) is an enzyme that catalyzes the formation of N-carbamoyl aspartate from aspartate and carbamoyl phosphate.

The subunit composition of E. coli ATCase (300 kD) is c₆r₆, where c and r denote the catalytic and regulatory subunits, respectively. The catalytic subunits are organized in a complex with 3 regulatory dimers (r₂) and two trimers (c₃). The activator ATP predominantly binds to ATCase's active state (R or high substrate affinity), while the inhibitor CTP predominantly binds to the enzyme's inactive state (T or low substrate affinity). The unreactive bisubstrate homolog N-(phosphonacetyl)-L-aspartate (PALA)binds firmly to R-state ATCase but not T-state ATCase. ATCase has an aspartate–binding domain and a carbamoyl phosphate–binding domain in each catalytic subunit.

PALA binds to the enzyme, which essentially mimics the binding of both the substrates, causing a conformational shift that swings the two domains together, allowing the two bound substrates to react and form the product. ATCase's T to R quaternary shift is triggered by conformational changes in a single catalytic subunit—movements of up to 8 Å for some residues. The enzyme's cooperative substrate binding is explained by the fact that binding substrate to one catalytic subunit enhances the substrate-binding affinity and catalytic activity of the other 5 catalytic .+

The activator ATP and inhibitor CTP attach to the same location on the regulatory subunit's outer edge, around 60 Å from the nearest catalytic site.CTP attaches to the T state preferentially, increasing its stability, while ATP binds to the R state preferentially, increasing its stability. The structural consequences of CTP and ATP binding to their less preferred enzyme states are also significant.

When CTP binds to the R-state of ATCase, it causes the regulatory dimer to contract, causing the catalytic trimers to move close together by 0.5 Å. As a result, critical residues in the enzyme's active sites are reoriented, reducing the enzyme's catalytic activity. When ATP binds to the T-state enzyme, it has the opposite effect: It makes the catalytic trimers shift by 0.4 Å, reorienting crucial residues in the enzyme's active sites and increasing catalytic activity.