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Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K(i) = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes., (© 2013 The Protein Society.)

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.

The amidotransferase or glutaminase domain (GLN domain) of mammalian carbamyl-phosphate synthetase II (CPSase II) catalyzes glutamine hydrolysis and transfers ammonia to the synthetase domain (CPS domain), where carbamyl phosphate formation is catalyzed in three consecutive reactions. The GLN and CPS domains are part of a single polypeptide and are connected via a 29-amino acid chain segment (GC linker). In contrast, the two comparable domains of Escherichia coli CPSase are not fused, but are separate, noncovalently associated subunits. To establish the function of the GC linker in mammalian CPSase, it was deleted, and the two domains were directly fused. The deletion mutant not only catalyzed glutamine-dependent carbamyl phosphate synthesis, but was activated 10-fold relative to its wild-type counterpart. However, ammonia-dependent synthesis of carbamyl phosphate was abolished, indicating that ammonia no longer had access to the active site on the CPS domain. The mutant was still sensitive to inhibition by the allosteric effector UTP, but was no longer activated by the allosteric effector phosphoribosyl pyrophosphate, although evidence indicated that the latter could bind to the enzyme. The linker appears to serve as a spacer that allows the complex to cycle between two conformations, an open low activity form in which the ammonia site on the CPS domain is accessible and an activated conformation in which the ammonia generated in situ from glutamine is directly channeled to the CPS active site and access to exogenous ammonia is blocked.

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.

The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.

Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway. DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests. A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain. Two recombinants were overexpressed in Escherichia coli. The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form. The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive. In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity. The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain. A 46-kDa soluble protein was expressed at 4% of the total soluble protein. The recombinant protein was catalytically active and had the expected amino-terminal sequence. The protein was purified to homogeneity. Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment. The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

CAD is a multidomain protein that catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The 243-kDa polypeptide consists of four functional domains; glutamine amidotransferase (GLNase), carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase). Controlled proteolysis of hamster CAD was found to cleave the molecule into 18 fragments which successively accumulate and disappear during the course of digestion. Each fragment was isolated and partially sequenced to determine its location in the polypeptide chain. Proteolysis was found to usually occur at the junctions between the domains and sub-domains identified by sequence homology. All proteases of low to moderate specificity cleaved the molecule in a similar fashion. The rate of proteolysis widely varied and the interdomain regions were not always accessible to proteases. Each of the major functional domains is postulated to consist of subdomains. The duplicated halves of the CPSase domain (116 kDa) have a homologous structure consisting of 11-, 25-26-, and 21-22-kDa subdomains. Prolonged digestion cleaved the DHOase domain (36.6 kDa) into two stable species suggesting that this region is comprised of 11.5- and 15.0-kDa subdomains. Similarly, proteolysis of the 21-kDa catalytic subdomain of the GLNase domain (40 kDa) indicated a bilobal structure consisting of 12.3- and 8.5-kDa chain segments. The connecting region between the two ATCase subdomains (16.4 and 18 kDa) was not cleaved. Copurification of many of the domains showed that they remain associated by noncovalent interactions even after the connecting segments have been cleaved. The chain segments, the linkers, which connect the domains and subdomains were conserved in length but not in sequence, were predicted to be relatively hydrophilic and flexible but did not show a tendency to assume a particular secondary structure. These studies provide a more detailed map of the structural organization of the CAD polypeptide.

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.

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.

The aspartate transcarbamylase activity of mammalian cells is carried by a large multifunctional protein, CAD, that catalyzes the first three steps in the de novo pyrimidine biosynthetic pathway. Controlled proteolysis of CAD cleaved the polypeptide chain into several separate structural domains which carried the individual activities of the complex. The aspartate transcarbamylase activity was associated with a 40,000-dalton proteolytic fragment, which kinetic studies showed was released in one of the first proteolytic cleavages. The species was purified to homogeneity by chromatography on carboxymethyl-Sephadex. The isolated species, designated the aspartate transcarbamylase domain, had a molecular weight under denaturing conditions of 40,000 +/- 1,500, a pI = 9.4, and a sedimentation coefficient (S20,w) of 6.2. The sedimentation coefficient suggested that the isolated domain was an oligomer consisting of two or three identical copies of the 40,000-dalton proteolytic fragment. The aspartate saturation curve obtained at a saturating concentration of carbamyl phosphate gave Km = 2.1 X 10(-2)M and Vmax = 119.5 mumol/min/mg, corresponding to a turnover number of 4,780 min-1. Like the aspartate transcarbamylase activity of CAD, the activity was strongly inhibited by high concentrations of aspartate. The corresponding parameters from the carbamyl phosphate saturation curve were Km = 2.07 X 10(-5)M, Vmax = 52.5 mumol/min/mg, and a turnover number of 2,153 min-1. The similarity of these parameters to those obtained from a steady state kinetic study of CAD indicated that the tertiary structure of this region of the polypeptide chain was largely preserved in the isolated species. In the absence of stabilizing agents, the half-life of aspartate transcarbamylase activity of CAD was 60.2 h, while that of the isolated domain was 10.6 h. This result suggested that there were interactions with other regions of the molecule which stabilized the structure of the aspartate transcarbamylase domain in the intact complex.

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)

The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240-kDa poly-peptide 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 composed of multiples of three copies of the CAD polypeptide chain. However, quantitative chemical crosslinking studies of CAD with dimethyl suberimidate were used here to show a progressive conversion of monomer to crosslinked hexamer. High levels of the hexamer accumulate in the reaction mixture, suggesting that the major oligomeric form is hexameric, although residual amounts of smaller oligomers remain present. Larger oligomers may form by association of hexamers and are seen after longer crosslinking times. Sucrose gradient centrifugation shows a 20.8S species to be the slowest sedimenting peak, while the larger species sediments at 27.9S. Electron microscopic studies of rotary-shadowed preparations of CAD have confirmed that, while small amounts of other oligomeric forms are present, the CAD monomer is primarily associated into cyclic hexamers with an open planar appearance.

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.

The multifunctional protein that initiates de novo pyrimidine biosynthesis in mammalian cells carries carbamoylphosphate synthetase, aspartate transcarbamylase (aspartate carbamoyltransferase), and dihydro-orotase activities on a single 215,000-dalton polypeptide chain. Kinetic studies of the controlled proteolysis of the molecule by elastase showed that the protein was not attacked at random by the protease but rather was successively cleaved into at least six well-defined proteolytic fragments. The initial cleavage converted the intact molecule into a 190,000-dalton species which appeared to retain all of the catalytic and regulatory functions of the native protein. This species was subsequently cleaved into two fragments, 150,000 and 40,000 daltons. The 40,000-dalton species, which carried the aspartate transcarbamylase activity, was resistant to further proteolysis; the 150,000-dalton polypeptide, which carried carbamoyl-phosphate synthetase and dihydro-orotase activities, underwent further digestion to 140,000 daltons. Continued proteolysis produced two species, 79,000 and 45,000 daltons; like the 40,000-dalton species, these were stable against further elastase digestion. The aspartate transcarbamylase and dihydro-orotase activities and the regulatory functions were preserved throughout the course of digestion; the carbamoylphosphate synthetase activity was more labile. By using sucrose gradient centrifugation and ion exchange chromatography, the 40,000- and 45,000-dalton species have been isolated. The 40,000-dalton fragment was found to have only aspartate transcarbamylase activity; the 45,000-dalton fragment has only dihydro-orotase activity. These experiments showed that this multifunctional protein is organized as discrete structural domains in which regions of the polypeptide chain are autonomously folded into separate functional units.

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.