Glutamine domain of the chimeric protein, CAD, that initiates pyrimidine biosynthesis in mammalian cells

Glutamine dependent carbamyl phosphate synthesis, the first step in mammalian de novo pyrimidine biosynthesis, is catalyzed by a 240 kDa chimeric protein, CAD, that also has the aspartate transcarbamylase and dihydroorotase activities. The complex was found to have a separate glutaminase activity of 0.04 μmol/min/mg, that increased five fold in the presence of bicarbonate and ATP. To determine whether the glutaminase activity, which provides ammonia for carbamyl phosphate synthesis, is associated with a separate structural domain (GLN), CAD was subjected to controlled proteolysis with elastase. The glutaminase, glutamine and ammonia dependent carbamyl phosphate synthetase activities, as well as the partial reactions; carbamyl phosphate dependent ATP synthesis and bicarbonate dependent ATPase, were correlated with the concentration of the various proteolytic fragments that accumulated in the digest. While the glutamine dependent carbamyl phosphate synthetase was rapidly inactivated, the glutaminase activity was found to be very resistant to proteolysis. The glutamine binding site of CAD was also specifically modified with 6-diazo-5-oxo-L-norleucine (DON). The modification was accompanied by a loss of both glutaminase and glutamine dependent carbamyl phosphate synthetase activities. Bicarbonate and ATP increased the rate of reaction of CAD with DON, while glutamine protected against inactivation. The stoichiometry of the reaction and the identity of the modified proteolytic fragments was determined using 14C labelled DON