The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240.kDa polypeptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers com (3), while a distinct 44-kDa species, the DHOase domain, catalyzes the DHOase reaction (4).The protein is an oligomer composed of multiple copies of the 240-kDa polypeptide. Trimers, hexamers, nonamers, and higher oligomeric forms have been reported (1). These authors found that the predominant form is the trimer, and that this species is in slow equilibrium with lower concentrations of hexamer and much smaller, but significant amounts, of the larger oligomers. All of these species are composed of multiples of three polypeptides, suggesting that the trimer is the fundamental oligomeric unit. There is evidence (1, 5) that the oligomeric structure is extremely sensitive to proteases and that nicking the polypeptide results in a marked reduction in the molecular weight of the protomer.We have examined the oligomeric structure of CAD prepared under conditions that eliminate nicking of the polypeptide chain (6, 7 MA) and removed from the surface by incubation in 0.5 mM EDTA in phosphate-buffered saline at 370C for 5 min. CAD was isolated by the method of Coleman et al. (1) and stored at -700C at a concentration of 3 mg/ml in 20 mM Tris/50 mM KCI/4 mM glutamine/4 mM aspartate/0.1 mM EDTA/1 mM dithiothreitol/5% glycerol/30% dimethyl sulfoxide, pH 7.4.Chemical Crosslinking. The crosslinking reaction was initiated by the addition of dimethyl suberimidate (Pierce) to CAD in storage buffer to give a final concentration of 0.3 mg of dimethyl suberimidate and 0.2 mg of CAD per ml (1, 9). The pH of the reaction mixture was 9.2. The reaction was carried out at room temperature, and aliquots were removed at timed intervals from 10 to 180 min and overnight. The crosslinking was quenched by the addition of 1 M glycine to a concentration of 0.1 M. The crosslinked species were analyzed by electrophoresis on NaDodSO4/composite 2.2% acrylamide-0.5% agarose tube gels (1, 10) with the Weber and Osborn (11) phosphate buffer system. Electrophoresis was carried out for 7 hr at a constant current of 8 mA per tube. The gels were stained with Coomassie blue and scanned with a Helena Laboratories (Beaumont, TX) Quick Scan densitometer.Sucrose Density Centrifugation of CAD. The buffer exchange method described by Penefsky (12) was used to remove the dimethyl sulfoxide and glycerol from CAD samples used for sucrose gradient centrifugation. The protein at 2.0-2.5 mg/ml (0.20 ml; 0.4-0.5 mg in 0.05 M Tris/0.05 M glycine, pH 8.7) was layered onto 5.0 ml of 5-20% linear sucrose gradients (Schwarz/Mann density gradient grade) in 0.05 M Tris glycine (pH 8.7) and then centrifuged for 0.5-4 hr at 189,000 x g in a Beckman L5-65 ultracentrifuge (at ...

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.