Search

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein CAD, consisting of glutamine-dependent carbamylphosphate synthetase, aspartate transcarbamylase, and dihydroorotase. The intracellular distribution of CAD in two hamster cell lines, BHK 21 and BHK 165-23 (a strain in which the CAD gene was selectively amplified), was determined by differential centrifugation and by two different cytochemical immunolocalization methods. Ammonia-dependent carbamylphosphate synthetase I was found in both cell types at a concentration of 0.01% of the total cell protein, so its distribution was also determined as a control for possible cross-reactivity of the CAD antibody probes and as a mitochondrial marker. CAD was localized in the cytoplasmic compartment and almost completely excluded from the nucleus. A punctate staining pattern suggested that it was not uniformly dispersed throughout the cytosol (unlike typical soluble proteins) but was associated with subcellular organelles. Although there was a slight tendency for CAD to be localized in the vicinity of the nuclear envelope, the amount of staining was much less than expected from differential centrifugation, which showed that 30% of the protein was found in the nuclear fraction. No interactions with other subcellular components could be detected by centrifugation. It is possible, however, that CAD is associated with subcellular structures that cosediment with the nuclei. Despite a 150-fold increase in CAD concentration in the over-producing cells, the distribution of the protein was unaltered. CAD was not concentrated near the mitochondria where the next enzyme of the de novo pathway, dihydroorotate dehydrogenase, is localized, which indicates that the intermediate dihydroorotate is not channeled, but rather dissociates from CAD and diffuses through the bulk cellular fluid.