A RECIPROCAL ALLOSTERIC MECHANISM FOR EFFICIENT TRANSFER OF LABILE INTERMEDIATES BETWEEN ACTIVE-SITES IN CAD, THE MAMMALIAN PYRIMIDINE-BIOSYNTHETIC MULTIENZYME POLYPEPTIDE

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase ( CPS Il) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa mult ienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile i ntermediate between the two active sites, we have used assays that dif ferentiate the synthesis of carbamoyl phosphate from the overall react ion of CPS II and ATCase that produces carbamoyl aspartate. We provide d excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three feature s indicate interactions between the folded CPS II and ATCase domains c ausing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the asparta te concentration is high ATCase uses endogenous carbamoyl phosphate fo r the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled ca rbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. T hirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more acti ve P state, which is induced by high concentrations of ATP and magnesi um ions and when 5'-phosphoribosyl diphosphate (the allosteric activat or) is present with low concentrations of ATP; these are conditions th at would be met during active biosynthesis in the cell. We propose a p henomenon of reciprocal allostery that encourages the efficient transf er of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase cause s a conformational change at the active site of the liganded form of C PS II, which protects it from inhibition by its product, carbamoyl pho sphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous c arbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allos tery justifies the close association of the enzyme activities within t he polypeptide and ensures that carbamoyl phosphate is efficiently syn thesised and is dedicated to the second step of pyrimidine biosynthesi s. These conditions fulfill those required for metabolic channeling in the cell.