A Novel Carbamoyl Phosphate Synthetase from Aquifex aeolicus 1. Anupama Ahuja, Cristina Purcarea, Hedeel I. Guy and David R. Evans

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1 JBC Papers in Press. Published on September 26, 2001 as Manuscript M A Novel Carbamoyl Phosphate Synthetase from Aquifex aeolicus 1 Anupama Ahuja, Cristina Purcarea, Hedeel I. Guy and David R. Evans Department of Biochemistry and Molecular Biology Wayne State University School of Medicine Detroit, Michigan Phone: FAX: devans@cmb.biosci.wayne.edu Running title: Aquifex aeolicus carbamoyl phosphate synthetase 1

2 SUMMARY Aquifex aeolicus, an extreme hyperthermophile, has neither a full length carbamoyl phosphate synthetase (CPSase) resembling the enzyme found in all mesophilic organisms, nor a carbamate kinase like CPSase such as those present in several hyperthermophilic archaea. However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains. The GLN, CPS.A and CPS.B homologs from A. aeolicus were cloned, overexpressed in E. coli and purified to homogeneity. The isolated proteins could catalyze several partial reactions but not the overall synthesis of carbamoyl phosphate. However, a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate and ammonia. The inclusion of the GLN subunit resulted in the formation of a 171-kDa complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised. Molecular modeling, using E. coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that all the residues known to be essential for function are conserved and properly positioned. A steady state kinetic study at 78 o C indicated that while the substrate affinity was similar for bicarbonate, ammonia and glutamine, the K m for ATP was appreciably higher than that of any known CPSase. The A. aeolicus complex, with a split gene encoding the major synthetase domains and relatively inefficient coupling of amidotransferase and synthetase functions, may be more closely related to the ancestral precursor of contemporary mesophilic CPSases. -end of summary- 2

3 Carbamoyl phosphate is the initial intermediate in the biosynthesis of both pyrimidine and arginine in all organisms, and of urea in ureotelic species. In most mesophiles, carbamoyl phosphate synthetase (CPSase 2, EC ) catalyzes the reaction: glutamine + HCO ATP carbamoyl phosphate + 2 ADP + P i The enzyme from Escherichia coli consists of a 120-kDa synthetase subunit (CPS) and a 40-kDa amidotransferase or glutaminase subunit (GLN) (1). Upon dissociation of the heterodimer (2,3), the GLN subunit was found to catalyze the hydrolysis of glutamine while the CPS subunit catalyzed the synthesis of carbamoyl phosphate from ammonia, bicarbonate and ATP. The GLN and CPS domains are fused in CAD (4-6), a mammalian multifunctional protein that catalyzes the first three steps of the de novo pyrimidine biosynthetic pathway. While glutamine hydrolysis is the usual source of ammonia for carbamoyl phosphate synthesis, mitochondrial CPSase I, the enzyme that catalyzes the first step in the urea cycle (1), has an inactive homologue of the GLN subunit fused to the amino end of the synthetase subunit. Consequently, CPSase I cannot hydrolyze glutamine and instead directly uses ammonia as the nitrogen-donating substrate. Although the CPSases have a diverse structural organization, they share a common catalytic mechanism (1) that proceeds through a complex series of partial reactions: 1) HCO ATP carboxy phosphate + ADP 2) carboxy phosphate + NH 3 carbamate + P i 3) carbamate + ATP carbamoyl phosphate + ADP 3

4 Lusty and her associates (7,8) were the first to clone and sequence both subunits of a member of this family of enzymes, E. coli CPSase. They noted that the sequence of the synthetase subunit consists of two highly homologous halves that probably arose from a duplication, translocation and fusion of an ancestral kinase gene. Similarly, they found that the GLN subunit consists of two domains, one of which shared homology to other triad-type amidotransferases (9). This structural organization was confirmed when the x-ray structure of E. coli CPSase was solved (10). One of the most interesting aspects of the x-ray structure was the presence of intramolecular tunnels connecting the active sites that ensures that the labile intermediates are sequestered. In studies of several different CPSases (10-18), each of the two halves of the synthetase subunit, designated CPS.A and CPS.B, were found to have an ATP binding site and to catalyze different ATP-dependent partial reactions. Site-directed mutagenesis of the E. coli enzyme showed (15) that CPS.A catalyzes the activation of bicarbonate while CPS.B is responsible for the phosphorylation of carbamate to form carbamoyl phosphate. While the two domains have a specialized function in the native molecule, they are functionally equivalent (19,20) when separately cloned and expressed in E. coli. The isolated domains form homodimers that catalyze both ATP-dependent partial reactions and the overall synthesis of carbamoyl phosphate from bicarbonate, NH 3 and two ATP molecules. The only apparent functional difference between the CPS.A and CPS.B dimers is that the latter is subject to allosteric control because effectors bind to the regulatory subdomain (21-27) at the extreme carboxyl end of CPS.B. An entirely different strategy for the synthesis of carbamoyl phosphate may be employed by some hyperthermophilic archaebacteria. In P. abyssi (28,29) and P. furiosus (30,31), carbamoyl phosphate can be synthesized by the ATP-dependent phosphorylation of carbamate 4

5 formed spontaneously in solution from ammonia and bicarbonate. The sequence and structure (32) closely resembles the catabolic carbamate kinases found in several eubacterial species and they have been designated carbamate kinase-like CPSases. With the advent of genome sequence projects, it became apparent that some hyperthermophilic organisms, including Methanococcus jannaschii (33) and Aquifex aeolicus (34), do not have an enzyme homologous to the CPSases found in mesophilic organisms. Instead, these genomes have open reading frames that encode proteins with deduced amino acid sequences that closely resemble the GLN, CPS.A and CPS.B domains of mesophilic CPSases. We report, for the first time, the cloning and expression of these genes from A. aeolicus and the reconstitution of functional complexes. EXPERIMENTAL PROCEDURES Materials: Pfu DNA polymerase was obtained from Stratagene; DNA ligase was from GibcoBRL; sodium [ 14 C] bicarbonate from NEN Life Science Products, Inc; Sephacryl S-300 High Resolution was from Amersham Pharmacia; the nucleotides and the enzymes pyruvate kinase, lactate dehydrogenase, hexokinase, glucose 6-phosphate dehydrogenase and glutamate dehydrogenase were from Sigma. A. aeolicus aspartate transcarbamoylase (ATCase) was isolated 3 as described elsewhere. Strains and Plasmids: A. aeolicus chromosomal DNA was a generous gift of Drs. Karl O. Stetter and Robert Huber from Regensburg University. The E. coli strains used were DH5α (GibcoBRL) and BL21(DE3) (Stratagene). The plasmid, prsetb, a His-tag expression vector (Invitrogen) was used for the construction of paacpsa, paacpsb and paagln 5

6 recombinants, encoding the A. aeolicus CPS.A, CPS.B and glutaminase (GLN) subunits, respectively. Cloning and Expression: The genes encoding the carbamoyl phosphate synthetase subunits were obtained by PCR using Pfu DNA polymerase (Stratagene), 180 ng of the template A. aeolicus chromosomal DNA, and 100 pmoles of primers. The 5 and 3 primers used for amplification were for cara (GLN): 5'-CGGAACTCGAGCATTTTGGCGCTTGAGGACGG-3' and 5'-ACTTCTGCAGCTCCTC ATCCCTGAGCCAT-3', for carb2 (CPS.A): 5'-CTTTGGGAT CCAAAAGGACGGACATCAAG-3 and 5'-TAAACAAGATCTTTAATCTTCAT CAAGGATTTC-3' and for carb1 (CPS.B): 5'-TTCTTGGATCCTAAAAAGG TTGTAATACTCGGA-3' and 5'-ATAAAGATCTCTAGGTCCATAAGAATTTGTA-3'. Each primer also included a restriction site to facilitate subcloning: XhoI/PstI for cara, BamHI/BglII for carb1 and BamHI/BglII for carb2. The amplified DNA fragments were cleaved with the appropriate restriction enzymes and inserted into the corresponding sites of the prsetb expression vector and co-transformed with the plasmid psjs1240 into E. coli BL21 (DE3). The helper plasmid psjs1240 (35) encodes two trna synthetases that recognize the codons AUA for isoleucine and AGA for serine. These codons occur infrequently in E. coli but are highly represented in the A. aeolicus genome. Purification: Each of the proteins is expressed as a fusion protein with a 3-kDa His-tag polypeptide attached to the amino end of the enzyme. The cells harvested from a 100-ml culture were resuspended in 3 ml of 50 mm TrisHCl, ph 8, 10 mm 2-mercaptoethanol, and disrupted by six 30-s bursts of sonication. The cell-extract was centrifuged at 17,000 x g for 20 min and the 6

7 supernatant was applied to a 1.5-ml Ni ++ -Probond column pre-equilibrated with 50 mm TrisHCl, ph 8, 10 mm 2-mercaptoethanol, and 200 mm NaCl. The column was washed with 20 ml of the same buffer. The A. aeolicus enzymes were then eluted with 1 ml of increasing concentrations of imidazole up to 200 mm in this buffer. The 1-ml fractions were analyzed by electrophoresis on 12.5% SDS-polyacrylamide gels and those containing pure proteins were dialyzed at 4 o C against 50 mm potassium phosphate buffer, ph 8, and 10 mm 2-mercaptoethanol. The proteins were stored in the same buffer except that 10% glycerol was added to the GLN subunit storage buffer. Enzyme Assays: Glutaminase Assay: The glutaminase activity was measured by coupling the formation of L-glutamate to the production of α-ketoglutarate using L-glutamate dehydrogenase (15). The assay mixture, consisting of varying concentrations of glutamine in 50 mm potassium phosphate buffer, ph 8, was pre-equilibrated at 78 o C for 1.5 min before starting the reaction with 30 µg of the enzyme. The reaction was quenched after 1.5 min with 50 µl of 1 M HCl and then placed on ice for 15 min. The ph was neutralized by the addition of 270 µl of 1 M TrisHCl, ph 10. The sample was then added to 600 µl of 0.1 M TrisHCl, ph 8, 0.8 mm acetylpyridine adenine dinucleotide (APAD), and 20 units of L-glutamate dehydrogenase (GDH). The absorbance at 363 nm of reduced APAD was measured after a 45-min incubation at 25 o C and the concentration of reduced APAD was calculated from a standard curve. Carbamoyl Phosphate Synthetase Assay: For measuring the ammonia-dependent CPSase, the assay mixture consisted of 200 mm ammonium chloride, 50 mm sodium [ 14 C] bicarbonate (100, ,000 dpm/µmol), 30 mm ATP, 32 mm MgCl 2, 100 mm KCl, 50 mm potassium phosphate, ph 8, 3 µg of purified A. aeolicus ATCase, 6 mm aspartate and 30 µg of the enzyme (unless specified otherwise) in a total volume of 0.5 ml. The assay mixture was the same for the 7

8 glutamine-dependent CPSase except that the ammonium chloride was replaced with 2 mm glutamine. The assay mixture without enzyme and bicarbonate was equilibrated at 78 o C for 1.5 min. The reaction was initiated by the addition of enzyme and bicarbonate and allowed to proceed for 1.5 min and quenched by the addition of 0.5 ml of 10% trichloroacetic acid. The samples were processed for counting as described previously (36). The saturation curves were obtained by varying the concentration of one substrate while fixing the others at the saturating concentrations given above. Partial Reactions: The bicarbonate-dependent ATPase was assayed by measuring the rate of ADP formation in the presence and absence of ammonium chloride using a pyruvate kinase/lactate dehydrogenase coupled assay (15). The assay mixture contained 50 mm bicarbonate, 100 mm KCl, 30 mm ATP, 32 mm MgCl 2, 10 mm 2-mercaptoethanol and, when present, 200 mm ammonium chloride in 50 mm potassium phosphate, ph 8, with the coupling substrates and enzymes. Carbamoyl phosphate-dependent ATP synthetase activity was assayed similarly by measuring the rate of ATP formation from ADP and carbamoyl phosphate, using a hexokinase/glucose-6-phosphate dehydrogenase coupled assay (15). The partial reactions were only assayed at 25 o C because of the limited stability of the coupling enzymes at elevated temperatures. Gel Filtration Chromatography: To determine whether A. aeolicus proteins form stable complexes, 0.5 ml aliquots (3-4 mg) of the purified proteins were applied to a 1.5 X 62-cm Sephacryl S-300 High Resolution column equilibrated with 50 mm potassium phosphate buffer, ph 8, 200 mm sodium chloride and 10 mm 2-mercaptoethanol. The column was eluted with the same buffer at a flow rate of 0.36 ml/min and fractions (0.5 ml) were analyzed by measuring the 8

9 absorbance at 280 nm, SDS-gel electrophoresis, and CPSase assays. Bovine serum albumin (66 kda), alcohol dehydrogenase (150 kda), apoferritin (443 kda) and blue dextran were used to calibrate the column. Data Analysis: The kinetic parameters were obtained by least squares fit of substrate saturation curves to the Michaelis-Menten or Hill equations, using the program Scientist (Micromath). Sequence comparison and multiple alignments were performed by using the GAP and Pileup programs of the GCG software package (37). BLAST software was used to search the A. aeolicus genome for open reading frames encoding homologs of mesophilic enzymes. Modeling of the three dimensional structure and energy minimization were performed using the program SwissModel (38-40) from ExPaSy server. The structure was visualized and analyzed using the Rasmol and Swiss PDB Viewer 3.7. RESULTS Identification and sequence analysis of A. aeolicus CPSase An exhaustive search of the Aquifex aeolicus genome failed to reveal any open reading frame encoding a protein homologous to the 120-kDa synthetase subunit of E. coli and other eubacterial CPSases. Similarly, a small 35-kDa carbamate kinase-like CPSase, responsible for carbamoyl phosphate synthesis in some archaeal species, was not detected when the genome was searched with seven query sequences representing highly conserved segments of carbamate kinases. However, two open reading frames encoding proteins homologous to the major structural domains of E. coli CPSase had been identified (34). The two A. aeolicus proteins 9

10 exhibited 39.5% sequence identity to each other (Fig. 1C), a value similar to that obtained ( %) when the two halves of mesophilic CPSases were aligned. One of these genes, designated carb2 (CPS.A in Fig. 1C) was found to have significantly greater sequence similarity to the CPS.A domain ( %) than the CPS.B domain ( %) of other CPSases. The converse was true for the second gene, carb1 (CPS.B), that was located 652 kb upstream of carb2 (Fig. 1A). Thus, the proteins encoded by the A. aeolicus carb2 and carb1 were designated CPS.A and CPS.B, respectively. An alignment of the A. aeolicus CPS.A and CPS.B subunits at the junction between the two fused domains in the E. coli enzyme is shown in Fig. 1B. Similarly, the cara gene could be unambiguously identified by its strong sequence similarity ( % identity) to the amidotransferase or glutaminase domain of other well characterized prokaryotic CPSases. CarA is located about 543 kb upstream of carb1, and overlaps pyrb, the gene encoding ATCase, by 3 base pairs. PyrC, which encodes dihydroorotase, was located approximately midway between cara and carb1 (Fig. 1). Expression and purification of the recombinant proteins The genes encoding CPS.A, CPS.B and GLN were amplified by PCR using A. aeolicus chromosomal DNA as the template. The oligonucleotides incorporated convenient restriction sites that allowed each DNA fragment to be inserted in frame into the prsetb expression vector. This vector appends a His-tag to the amino end of the recombinant protein to facilitate purification. The resulting constructs, paagln, paacpsa and paacpsb, encoding GLN, CPS.A and CPS.B, respectively, were sequenced to confirm the fidelity of the amplification process. Co-transformation of the E. coli BL21(DE3) strain with the helper plasmid psjs1240 resulted in the expression of high levels of the soluble proteins, 28, 40, and 30 mg per liter of 10

11 culture for the CPS.A, CPS.B and GLN, respectively. Each protein could be purified (Fig. 2) to homogeneity by Ni ++ affinity chromatography. Catalytic activity of the recombinant proteins The CPS.A and CPS.B subunits had no detectable NH 3 -dependent CPSase activity. In contrast, the GLN domain hydrolyzed glutamine to glutamate and ammonia at a rate of 0.87 nmol/min/mg at 25 o C. While neither CPS.A nor CPS.B alone could catalyze the overall reaction, a stoichiometric mixture of CPS.A and CPS.B catalyzed the formation of carbamoyl phosphate from ammonia, ATP and bicarbonate at a rate of 5.7 nmol/min/mg (25 o C). Under the same conditions, the rate of ATP hydrolysis was 10.7 nmol/min/mg 4. The ratio of ATP consumed to carbamoyl phosphate formed of 1.9 is consistent with the 2:1 stoichiometry of the classical CPSase mechanism. The isolated CPS.A and CPS.B subunits were found to catalyze both ATP-dependent partial reactions as assayed by established procedures (Experimental Procedures). The first partial reaction, the activation of bicarbonate, was measured as a bicarbonate-dependent ATPase in the absence of a nitrogen-donating substrate. Equivalent amounts of CPS.A, CPS.B and CPS.A-CPS.B had a similar bicarbonate-dependent ATPase activity (Table I). When ammonia was present, allowing the overall reaction to proceed, the ATPase activity of CPS.A and CPS.B remained for the most part unchanged, but the rate of ATP hydrolysis by the CPS.A-CPS.B complex increased 3-fold to 41 nmol/min/mg. The second ATP-dependent partial reaction, the phosphorylation of carbamate, is assayed in the reverse direction as carbamoyl phosphate-dependent ATP synthesis (Experimental Procedures). Again, both CPS.A and CPS.B could catalyze this reaction (Table I), although it 11

12 was surprising that the rate of ATP formation by CPS.A was significantly higher than that observed for either CPS.B or the CPS.A-CPS.B complex. Oligomeric structure of the recombinant proteins SDS-gel electrophoresis on calibrated polyacrylamide gels (Fig. 2) showed that the molecular mass of the CPS.A, CPS.B and GLN proteins was 64 ± 1.2, 63 ± 1.6 and 45 ± 1.0 kda, respectively. These values are close to the sizes of 62,289, 60,037 and 41,682 as predicted from the deduced amino acid sequence when the 3-kDa His-tag on the recombinant proteins is taken into consideration. The size of each protein was determined under non-denaturing conditions by size-exclusion chromatography on a calibrated Sephacryl S-300 column (Fig. 3A). CPS.A eluted as a 66-kDa species, indicating that it is monomeric. In contrast, CPS.B was more heterogeneous. The major species had a molecular mass of 170 kda, but there was an appreciable fraction that eluted in the void volume, suggesting that the isolated CPS.B aggregates. These results were confirmed by electrophoresis on non-denaturing polyacrylamide gels (results not shown). The stoichiometric mixture of CPS.A and CPS.B eluted as a 124-kDa dimer, although there was a shoulder on the leading edge that may represent the presence of larger species. Assay of CPSase activity in the column fractions (Fig. 3A) showed that only the CPS.A-CPS.B dimer had catalytic activity. A mixture of equimolar amounts of CPS.A, CPS.B and GLN was found to elute as a single 171-kDa species that could catalyze glutamine-dependent carbamoyl phosphate synthesis (Fig. 3B). A complex consisting of one copy of each subunit would be expected to have a mass of 173 kda. SDS-polyacrylamide gel electrophoresis of the peak fractions (Fig. 3B, insert) showed that the molar ratio of CPS.A-CPS.B:GLN was 1.2 5, consistent with a species comprised 12

13 of equivalent molar amounts of CPS.A-CPS.B and GLN subunits. It is interesting that the major species visualized on the SDS gel had a mass of 170 kda when the samples were not heated at 100 o C prior to electrophoresis (data not shown), suggesting that the complex is difficult to dissociate even in strong detergents. Taken together, these results show that the GLN, CPS.A and CPS.B associate to form a stable 1:1:1 heterotrimer. Thermostability of CPS.A-CPS.B The thermostability of the CPS.A-CPS.B complex was assessed by preincubating the protein at increasing temperatures for 10 min, rapidly cooling the sample and assaying the CPSase activity at 37 o C. The activity remained unchanged between 25 o C and 60 o C and then abruptly increased 54% at 70 o C (Fig. 4). The precipitous loss of catalytic activity above 80 o C is indicative of thermal denaturation. The temperature at which half the maximum activity was lost is 81 o C. The GLN-CPS.A-CPS.B complex had appreciably greater thermostability showing no loss of activity up to 90 o C. Interestingly, the large increase in thermal stability at temperatures prior to thermal denaturation of CPS.A-CPS.B was not observed in the GLN-CPS.A-CPS.B complex. Steady state kinetics The catalytic activity of CPS.A-CPS.B was found to increase with increasing temperature from a value of 0.02 µmol/min/mg at 25 o C to 0.5 µmol/min/mg at 78 o C. Since A. aeolicus grows optimally at elevated temperature, the steady state kinetic parameters for CPS.A-CPS.B were measured at 78 o C, as close to the thermal denaturation temperature (Fig. 4) as was practically 13

14 possible. Saturation curves were obtained (Fig. 5A-C) with NH 4 Cl, NaHCO 3 or ATP as the variable substrate. While the NH 4 Cl and NaHCO 3 curves exhibited Michaelis-Menten kinetics, the ATP saturation curve was sigmoidal and was fit to the Hill equation (Hill coefficient 1.4). The kinetic parameters, summarized in Table II, indicate that the K m values for bicarbonate (7.9 mm) and ammonia (3.2 mm) 6 for the A. aeolicus complex were comparable to the values HCO - NH obtained (41) for the E. coli CPSase synthetase subunit (K m 3 = 10.8 mm, K m 3 = 5.2 mm). However, the K m for ATP was 6-fold higher than that of the E. coli subunit (K ATP m = 1.3 mm). The k cat of the A. aeolicus synthetase complex (1.6 s -1 ), determined from an ATP saturation curve, was appreciably lower than the value obtained for the E. coli CPSase synthetase subunit (7.3 s -1 ) (41). The isolated GLN subunit has a relatively high affinity for glutamine (K m = 58 µm), but the k cat is very low (9 x 10-3 s -1 ), indicating a poor catalytic efficiency (Table II). However, when the GLN subunit forms a complex with the CPS.A-CPS.B dimer, the rate of glutamine hydrolysis increases 18 fold (k cat = s -1 ). The increased rate is partially offset by a 12-fold increase in the K m for glutamine. The k cat and K m values for the GLN-CPS.A-CPS.B complex (Fig. 5D-F) using ammonia as the nitrogen donating substrate are similar (Table II) to the values observed for the isolated CPS.A-CPS.B dimer. However, a comparison of the k cat values for the heterotrimer (Table II) for the overall synthesis of carbamoyl phosphate indicates that the catalytic efficiency of the heterotrimer is 4-9 fold lower when glutamine, rather than ammonia, serves as the nitrogen donating substrate. 14

15 Allosteric regulation As for E. coli CPSase, UMP is an allosteric inhibitor and ornithine is an activator of A. aeolicus CPSase. When the ATP saturation curve (Fig. 6A) for CPS.A-CPS.B was determined in the presence of 2 mm UMP, the K m was found to increase 2.3-fold and the V max decreased 30% (Table III). Together these changes in the kinetic parameters resulted in a 3-fold decrease in the apparent second order rate constant, k cat /K ATP m. Ornithine had little effect on the K m for ATP but increased the V max 1.8-fold, giving a k cat /K m ATP that was about 2-fold higher than the corresponding value for the enzyme in the absence of allosteric ligands. UMP and ornithine had a more pronounced effect on the glutamine-dependent CPSase activity of GLN-CPS.A-CPS.B. The presence of UMP increased the K m 360% and decreased the V max 80%, while ornithine decreased the K m 40% and increased the V max 320%. Thus, effectors can alter the apparent second order rate constant for the glutamine-dependent CPSase activity over a 100-fold range (Table III). The effect of increasing concentrations of the allosteric ligands on the catalytic activity of CPS.A-CPS.B and GLN-CPS.A-CPS.B (Fig. 6B-C) showed that half-maximal activation occurs at 0.1 mm ornithine for CPS.A-CPS.B and at about 2 mm for GLN-CPS.A-CPS.B. Very low concentrations of UMP activate ammonia- and glutamine-dependent CPSase activity, but higher concentrations inhibit in a concentration-dependent manner. Half-maximal inhibition occurred at 0.5 and 0.05 mm UMP for CPS.A-CPS.B and GLN-CPS.A-CPS.B, respectively. The maximum ornithine activation and UMP inhibition was appreciably greater for GLN-CPS.A-CPS.B than for the complex lacking the GLN subunit. Other allosteric effectors known to modulate the catalytic activity of other CPSases, including UTP, IMP, PRPP, N-acetyl-L-glutamate, had no effect on the catalytic activity of A. aeolicus CPSase. 15

16 Molecular modeling of the A. aeolicus GLN, CPS.A and CPS.B subunits The high degree of sequence identity between E. coli CPSase and the GLN, CPS.A and CPS.B subunits of A. aeolicus (49%, 63% and 55% identity, respectively) makes the alignment of the proteins unambiguous. Compared to E. coli CPSase, the A. aeolicus CPS.A subunit is nine residues longer on the carboxyl end of the polypeptide and has three short deletions (340, and in the E. coli polypeptide) and a single one-residue insertion (between in the E. coli sequence). The A. aeolicus CPS.B subunit is four residues shorter on the amino end and has one additional residue on the carboxyl end. There are three insertions in the A. aeolicus CPS.B subunit, 13 residues (between 874 and 875 in the E. coli enzyme), 4 residues (between 885 and 886) and one residue (between 1056 and 1057). Similarly, the GLN subunit, which is 6 residues shorter at the carboxyl end and with only three deletions (residues 131, 263, in the E. coli enzyme) can be aligned with the E. coli GLN subunit sequence with a high degree of confidence. The tertiary structure of the A. aeolicus CPS.A-CPS.B and GLN subunits were modeled using the E. coli CPSase (pdb: 1BXR) x-ray structure (42) as the tertiary template. A superposition of the template and model structures showed that the backbone is virtually identical. There are few insertions and deletions, all of which lie on the surface of the molecule in unobtrusive locations that would not be expected to disrupt function. The additional five residues (Fig. 1B) at the junction between CPS.A and CPS.B are located in a region far from all of the active sites. The other major insertion of 13 residues in CPS.B is a loop with an unusual sequence consisting of 7 charged and 6 very hydrophobic residues. Although the conformation 16

17 of this loop was not modeled, it is clearly located on the CPS.B surface in what appears to be a non-critical region. An examination of the model (Table IV) showed that all of the residues involved in substrate binding and catalysis in both the GLN and CPS subunits of the E. coli enzyme are conserved and properly positioned (42,43), as are all of the residues that constitute the proposed intermolecular tunnels (10) between the GLN-CPS.A domains and between CPS.A and CPS.B. The residues at the interdomain interfaces (Table V) are also conserved. All of the E. coli residues of CPS.B in contact with CPS.A, as well as those of CPS.A in contact with CPS.B, are identical or conservative substitutions in the A. aeolicus enzyme. An analysis of the residues of CPS.A, in the A. aeolicus CPSase model, that lie within 3Å of CPS.B (Table V) indicate that, of 24 interdomain contacts found in the E. coli enzyme, 17 are present in the A. aeolicus model (14 identical residues). Of 20 residues on CPS.B in close proximity to the CPS.A domain in the E. coli enzyme, 17 are found in the model structure. An analysis of the interactions between the GLN subunit and CPS.A of the model indicated that this intersubunit interface was also similar. Thus, it is likely that the intersubunit interactions and arrangement of the A. aeolicus CPSase subunits are the same as in E. coli CPSase. DISCUSSION A functional Aquifex aeolicus CPSase was assembled from three subunits homologous to the CPSase domains of other organisms. The isolated CPS.A and CPS.B subunits catalyze the partial reactions, but ammonia-dependent carbamoyl phosphate synthesis requires the concerted action of both subunits. The addition of the GLN subunit confers the ability to utilize glutamine 17

18 as a nitrogen-donating substrate. The complex is a 171-kDa heterotrimer composed of one copy of each type of subunit. Sequence comparisons and molecular modeling indicate that the structure of the A. aeolicus heterotrimer closely resembles the enzyme from E. coli and other mesophilic organisms. In principle, it is possible that the enzyme catalyzes carbamoyl phosphate synthesis via a carbamate kinase-like mechanism, from carbamate formed spontaneously in solution from ammonia and bicarbonate. This has been documented (28-31) in some hyperthermophilic archaebacterial enzymes. However, there are several lines of evidence that argue conclusively against this possibility, 1) the CPS.A and CPS.B subunits catalyze both ATP-dependent partial reactions, 2) the stoichiometry of the reaction, in accord with the classical mechanism (reactions 1-3), is two moles of ATP consumed per mole of carbamoyl phosphate synthesized and 3) the complete complex catalyzes the glutamine-dependent CPSase reaction in the absence of ammonia, an observation that is difficult to reconcile with a carbamate kinase-like mechanism. Thus, we conclude that carbamoyl phosphate is synthesized in A. aeolicus by the classical mechanism involving the concerted action of two ATP binding sites that catalyze a complex series of sequential partial reactions. The intramolecular tunnels, a pre-requisite for sequestering the unstable intermediates are present in the model structure. While the overall turnover number is low, the other steady state kinetic parameters of the A. aeolicus complex are close to the values reported for E. coli CPSase. The only notable exceptions are 1) the K m for ATP is unusually high in both the CPS.A-CPS.B and the GLN- CPS.A-CPS.B complexes, and 2) the k cat for glutamine-dependent carbamoyl phosphate synthesis is about seven fold lower than the value obtained using ammonium chloride as the 18

19 nitrogen source. Thus, unlike the situation in other CPSases (44,45), the rate of the reaction appears to be limited by the rate of glutamine hydrolysis. The coupling between glutamine hydrolysis and the activation of bicarbonate (reaction 1) is an important aspect of the coordination of these parallel reactions in glutamine-dependent carbamoyl phosphate synthesis. The reactions must occur in phase to avoid the wasteful hydrolysis of glutamine and ATP. The isolated CAD GLN domain, obtained by cloning and expression, has a very low k cat and a very high K m for glutamine (45,46). When combined with the CPS domain, the k cat for glutamine hydrolysis increases 17-fold and the K m decreases 47- fold, indicating that interdomain interactions appreciably improve the catalytic efficiency of glutamine hydrolysis. The k cat /K m was found to increases 800% upon association. In the case of the isolated A. aeolicus GLN subunit, the k cat is low (Table II) but the K m is also low indicating high affinity for the substrate. When associated with CPS.A-CPS.B, the k cat increases 18-fold, but the affinity for glutamine is 12-fold lower, so the apparent second order rate constant increases by only 50%. In E. coli and mammalian CPSases, the activity of the GLN domain is very low in the absence of the other substrates needed for carbamoyl phosphate synthesis, but the k cat increases fold when these substrates bind to the synthetase domain (1,47,48). This coupling mechanism is thought to ensure that reactions occurring on distinct domains remain in phase. Preliminary experiments with the A. aeolicus GLN-CPS.A-CPS.B complex indicated that the presence of ATP and bicarbonate stimulate the glutaminase activity less than two fold (unpublished), indicating that the coupling mechanism is relatively inefficient in the A. aeolicus complex. 19

20 There is good evidence (11-18) that CPS.A and CPS.B in the native complex have specialized functions. CPS.A catalyzes the activation of bicarbonate (reaction 1), while CPS.B phosphorylates carbamate (reaction 3). However, the isolated CPS.A and CPS.B subdomains of mammalian, yeast and E. coli CPSase were found (19,20) to form homodimers that could catalyze ammonia-dependent carbamoyl phosphate synthesis. If the homodimer was dissociated by high pressure (49), the monomers reversibly lost CPSase activity but retained the ability to catalyze both partial reactions. Similarly, the isolated A. aeolicus CPS.A and CPS.B subunits each effectively catalyzed both partial reactions (Table I). This result is not unexpected given the - similarity of the reactions catalyzed and the isosteric nature of the substrates, HCO 3 and carbamate. While the isolated A. aeolicus subunits catalyze the partial reactions, they do not catalyze the overall synthesis of carbamoyl phosphate, probably because they cannot form functional dimers. The CPS.A subunit is a monomer, while CPS.B tends to aggregate. The failure of the subunits to dimerize is a necessary constraint for this type of molecular organization. Since CPS.A and CPS.B are expressed as individual polypeptides that must be assembled in vivo, if functional homodimers could form, a substantial fraction of the complexes, 25%, would be of the type (CPS.A) 2 and would thus be unregulated. The relative rates of the partial reactions catalyzed by the A. aeolicus CPS.A and CPS.B subunits do not agree with the functions assigned to these domains in other CPSases. CPS.A catalyzes carbamoyl phosphate-dependent ATP synthesis three times faster than does CPS.B. A similar trend is observed for the bicarbonate-dependent ATPase, although the difference is not as large. One might argue on this basis that our assignment is wrong and that the subunit that we have designated CPS.B is in reality CPS.A and vice versa. However, comparison of the CPSase 20

21 sequences (Fig. 1) strongly suggests that this explanation is incorrect. Experiments are planned to directly assess the function of the two domains in the intact A. aeolicus complex. In E. coli, the CPS.A and CPS.B domains are fused to form a single synthetase subunit, while the genes encoding the CPS and GLN subunits are part of an operon that ensures their coordinated expression. In contrast, the A. aeolicus genes encoding the CPSase subunits are scattered throughout the genome. Moreover, the individual subunits fold independently into functional units capable of catalyzing the partial reactions and, thus, unproductive futile cycles would occur unless the individual subunits are rapidly incorporated into a fully assembled complex. Consequently, coordinated regulation of the expression of these genes must be a major factor in ensuring that stoichiometric amounts of the CPSase subunits are simultaneously synthesized. An examination of 200 bp of the sequences upstream of these genes did not reveal any common transcription factor binding sites, but a detailed study of the regulation of the expression of the subunits would be informative. One especially interesting aspect of the organization of the pyrimidine biosynthetic genes in A. aeolicus is that the ATCase and GLN coding sequences overlap. This gene arrangement may suggest the coordinated expression of the GLN, CPS and ATCase genes, a potentially important control mechanism since A. aeolicus CPSase functions in concert with ATCase to facilitate channeling 3 of the unstable carbamoyl phosphate, thus, preserving it from thermal degradation. Mesophilic CPSases are thought (7,50-52) to have arisen by a gene duplication, translocation and fusion of an ancestral kinase followed by the acquisition of an amidotransferase subunit that conferred the ability to utilize glutamine. A. aeolicus CPSase with split genes encoding separate CPS.A and CPS.B subunits and relatively inefficient coupling of 21

22 amidotransferase and synthetase functions may be more closely related to the ancestral precursor of contemporary mesophilic CPSases. Acknowledgements: We would like to thank J. Andrew Berkowski for expert technical assistance and Dr. J. Sandler for the generous gift of the plasmid psjs1240. REFERENCES 1. Meister, A. (1989) Adv. Enzymol. Relat. Areas. Mol. Biol. 62, Trotta, P. P., Burt, M. E., Haschemeyer, R. H., and Meister, A. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, Trotta, P. P., Pinkus, L. M., Haschemeyer, R. H., and Meister, A. (1974) J. Biol. Chem. 249, Hoogenraad, N., Levine, R., and Kretchmer, N. (1971) Biochem. Biophys. Res. Commun. 44, Shoaf, W. T., and Jones, M. E. (1973) Biochemistry 12, Coleman, P., Suttle, D., and Stark, G. (1977) J. Biol. Chem. 252, Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, Nyunoya, H., and Lusty, C. J. (1984) J. Biol. Chem. 259, Zalkin, H. (1985) Methods Enzymol. 113, Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M., and Rayment, I. (1997) Biochemistry 36, Rubio, V., Britton, H. G., and Grisolia, S. (1979) Eur. J. Biochem. 93, Britton, H. G., Rubio, V., and Grisolia, S. (1979) Eur. J. Biochem. 102,

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24 30. Durbecq, V., Legrain, C., Roovers, M., Pierard, A., and Glansdorff, N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, Marina, A., Alzari, P. M., Bravo, J., Uriarte, M., Barcelona, B., Fita, I., and Rubio, V. (1999) Protein Sci. 8, Ramon-Maiques, S., Marina, A., Uriarte, M., Fita, I., and Rubio, V. (2000) J. Mol. Biol. 299, Bult, C. J., White, O., Olsen, G. J., Zhou, L., Fleischmann, R. D., Sutton, G. G., Blake, J. A., FitzGerald, L. M., Clayton, R. A., Gocayne, J. D., Kerlavage, A. R., Dougherty, B. A., Tomb, J. F., Adams, M. D., Reich, C. I., Overbeek, R., Kirkness, E. F., Weinstock, K. G., Merrick, J. M., Glodek, A., Scott, J. L., Geoghagen, N., and Venter, J. C. (1996) Science 273, Deckert, G., Warren, P., Gaasterland, T., Young, W., Lenox, A., Graham, D., Overbeek, R., Snead, M., Keller, M., Aujay, M., Huber, R., Feldman, R., Short, J., Olson, G., and Swanson, R. (1998) Nature 392, Kim, R., Sandler, J., Goldman, S., Yokota, H., Clark, A. J., and Kim, S. H. (1998) Biotechnology Letters 20, Guy, H. I., and Evans, D. R. (1995) J. Biol. Chem. 270, Needleman, S. B., and Wunsch, C. D. (1970) J. Mol. Biol. 48, Peitsch, M. C. (1995) Biotechnology 13, Peitsch, M. C., Wells, T. N., Stampf, D. R., and Sussman, J. L. (1995) Trans. Biochem. Sci. 20, Peitsch, M. C. (1996) Biochem. Soc. Trans. 24, Rubino, S. D., Nyunoya, H., and Lusty, C. J. (1987) J. Biol. Chem. 262,

25 42. Thoden, J. B., Wesenberg, G., Raushel, F. M., and Holden, H. M. (1999) Biochemistry 38, Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, Raushel, F. M., and Villafranca, J. J. (1979) Biochemistry 18, Hewagama, A., Guy, H. I., Chaparian, M., and Evans, D. R. (1998) Biochim. Biophys. Acta. 1388, Guy, H. I., and Evans, D. R. (1994) J. Biol. Chem. 269, Chaparian, M. G., and Evans, D. R. (1991) J. Biol. Chem. 266, Lusty, C. J., and Liao, M. (1993) Biochemistry 32, Guy, H. I., Schmitt, B., Hervé, G., and Evans, D. R. (1998) J. Biol. Chem. 273, Nyunoya, H., Broglie, K. E., and Lusty, C. J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, Rubio, V., Cervera, J., Lusty, C. J., Bendala, E., and Britton, H. G. (1991) Biochemistry 30, Davidson, J., Chen, K., Jamison, R., Musmanno, L., and Kern, C. (1993) Bioessays 15,

26 FOOTNOTES 1 This research was supported by a grant (MCB ) from the National Science Foundation and NIH grant (GM/CA60371). 2 Abbreviations used are: CPSase, carbamoyl phosphate synthetase or its activity; CPS, the synthetase subunit-composed of CPS.A and CPS.B in most organisms; CPS.A, the CPSase synthetase subunit encoded by carb2 as designated in Genbank; CPS.B, the CPSase synthetase subunit encoded by carb1 as designated in Genbank; GLN, glutaminase subunit encoded by cara; ATCase, aspartate transcarbamoylase; DHOase, dihydroorotase; CAD; the mammalian protein composed of Gln, CPS, ATCase and DHOase; APAD, acetyl pyridine adenine dinucleotide; GDH, L-glutamate dehydrogenase; PRPP, phosphoribosyl 5 -pyrophosphate. 3 Purcarea, C., Lu, T., Ahuja, A., Kovari, L., and Evans, D. R. (2001) J. Biol. Chem. manuscript in revision. 4 The initial assays were conducted with a fixed ATP concentration of 10 mm and thus, the values obtained were lower than those in Table I measured in the presence of 30 mm ATP. 5 While the difference in mobility between CPS.A and CPS.B can be visualized on the SDS gel, the resolution was not sufficient to individually quantitate each species. 6 The K m for ammonia was calculated from the value in Table II for ammonium chloride assuming a pk a of 8.88 for the dissociation of the proton from the ammonium ion (38). 26

27 Table I. ATP-dependent partial reactions a HCO 3 - -dependent-atpase CP-dependent Protein ATP synthetase -NH 3 +NH 3 nmol/min/mg nmol/min/mg nmol/min/mg CPS.A 9.62 ± ± ± 0.04 CPS.B 11.0 ± ± ± 0.04 CPS.A-CPS.B b 14.5 ± ± ± 0.04 a All assays were conducted at 25 o C. The formation of ADP or ATP was determined in coupled enzyme assays as described in the Experimental Procedures. b For the CPS.A and CPS.B assays, 60 µg of each protein was present in the assay mixture. For the CPS.A-CPS.B assays, 30 µg of each subunit was used. 27

28 Table II. Steady state kinetic parameters a Variable Nitrogen Substrate Substrate K m V max k cat CPS.A-CPS.B mm µmol/min/mg s -1 ATP b NH 4 Cl 7.43 ± ± ± 0.05 HCO 3 - NH 4 Cl 7.92 ± ± ± 0.04 NH 4 Cl NH 4 Cl 28.1 ± ± ± 0.02 GLN Glutamine Glutamine ± ± ± GLN-CPS.A-CPS.B ATP Glutamine 6.88 ± ± ± ATP b NH 4 Cl 5.08 ± ± ± HCO 3 Glutamine 2.73 ± ± ± HCO 3 NH 4 Cl 13.4 ± ± ± Glutamine Glutamine ± ± ± NH 4 Cl NH 4 Cl 35.7 ± ± ± a CPSase activity measured at 78 o C. b The ATP saturation curve is somewhat sigmoidal and fit the Hill equation better than the Michaelis-Menten equation. In this analysis the Km = [S] 0.5 and the Hill coefficient n = 1.38 for CPS.A-CPS.B and n = 2.06 for GLN-CPS.A-CPS.B. All other data was fit to the Michaelis- Menten equation. 28

29 Table III. Allosteric regulation Effector K m V max k cat k cat /K m mm µmol/min/mg s -1 M -1 s -1 CPS.A-CPS.B None a 8.1 ± ± ± UMP (2 mm) 18.0 ± ± ± Ornithine (5 mm) 7.8 ± ± ± GLN-CPS.A-CPS.B None a 10.1 ± ± ± UMP (2 mm) Ornithine (5 mm) 6.33 ± ± ± a data fit to the Hill equation. The Hill coefficient obtained was 1.5 ± 0.2 for both CPS.A-CPS.B and GLN-CPS.A-CPS.B. All other saturation curves were fit to the Michaelis-Menten equation. 29

30 Table IV. Catalytic and tunnel residues Active Site Residues Ammonia Tunnel Carbamate Tunnel GLN CPS.A CPS.B GLN CPS.A CPS.A CPS.B Eco Aaeo Eco Aaeo Eco Aaeo Eco Aaeo Eco Aaeo Eco Aaeo Eco Aaeo S47 S47 R129 R129 R715 R158 D45 D45 S233 A233 E604 E47 G575 G18 G241 G237 R169 R169 G721 G164 K202 K198 I234 I234 E217 E217 E577 E20 G243 G239 G175 G175 G722 G165 H353 H348 A251 A251 I18 I18 R848 R291 C269 C264 G176 G176 D753 D196 S35 S35 Y261 Y261 V19 V19 K891 K350 Q273 Q268 E208 K208 b H754 K197 b M36 M36 N283 N283 I20 I20 E916 E374 G313 N308 a L210 L210 L756 L199 G293 G288 N301 N301 G21 G21 Q829 Q272 F314 F309 E215 E215 E761 E204 A309 A304 S307 S307 Q22 Q22 N843 N286 H353 H348 G241 G241 G786 G229 N311 N306 L310 L310 A23 A23 T849 T292 E355 E350 Q285 Q285 Q829 Q272 P358 P35 A311 A311 M174 L174 S913 S371 E299 E299 E841 E284 G359 G354 A314 A314 G175 G175 T914 T372 N301 N301 N843 N286 T315 T315 C232 C232 R306 R306 R848 R291 I352 I351 M378 M377 V381 V380 30

31 Table V. Close contacts between CPS.A and CPS.B domains a CPS.A CPS.B E. coli A. aeolicus E. coli A. aeolicus I18 (I) b I18 (I) K4 (K) P51 (P) P51 (P) Y23 (Y) T53 (T) T53 (T) I572 (I) T56 (T) T56 (T) H584 (H) H27 (H) D57 (D) D57 (D) P603 (P) P46 (P) P58 (P) T605 (T) T48 (T) E59 (E) E59 (E) T608 (T) T51 (T) T64 (T) D611 (D) D54 (D) E67 (E) D57 (D) P68 (P) R615 (K) K58 (R) D72 (E) L616 (L) L59 (L) K76 (E) F618 (F) F61 (F) G175 (G) E619 (E) E62 (E) S177 (T) T177 (S) I64 (V) E197 (D) E624 (E) E67 (E) K313 (K) K313 (K) H68 (D) K321 (K) K321 (K) E628 (D) N363 (D) T646 (T) T89 (T) K366 (K) K365 (K) K855 (K) K298 (K) E368 (G) K863 (K) K306 (K) M377 (M) K899 (R) R358 (K) K397 (K) K396 (K) E361 (G) R399 (R) M911 (M) M370 (M) E403 (E) E402 (E) T914 (T) T373 (T) K493 (K) K487 (K) K930 (K) D499 (D) D493 (D) T1059 (E) N496 (K1) R517 (R) H523 (V) A541 (P) P535 (A) M543 (Y) Y537 (M) S545 (S) S539 (S) a Residues in CPS.A and CPS.B that are within 3 Å of residues in the other domain. b Letters in parenthesis are the residues occupying the corresponding position in the enzyme from the other species. 31

32 FIGURE LEGENDS Figure 1. Genome organization and sequence identities Panel A: The organization of the genes encoding the enzymes catalyzing the first three steps of the de novo pyrimidine biosynthetic pathway in A. aeolicus. The genes are represented by arrows showing the direction of transcription. The cara, carb1, carb2 genes encoding the GLN, CPS.B and CPS.A subunits of A. aeolicus CPSase are located at , , , respectively. PyrB ( ) codes for ATCase and pyrc ( ) for DHOase. Panel B: An alignment of A. aeolicus CPS.A and CPS.B at the junction between domains in the E. coli enzyme. The A. aeolicus CPS.A subunit is nine residues longer than the CPS.A domain of the E. coli enzyme while the A. aeolicus CPS.B subunit is four residue shorter than the E. coli CPS.B domain. Panel C: Percent sequence identity between the CPS.A and CPS.B domains (unshaded) and between like-domains (shaded) of A. aeolicus (Aaeo), E. coli (Eco), M. jannaschii (Mjan), B. subtilis (Bsub), P. aeruginosa (Paer) and hamster (ham) CPSase. The A. aeolicus carb1 and carb2 are designated CPS.B and CPS.A in the figure. Figure 2. Purification of the recombinant proteins Cell extracts from a 100-ml culture of E. coli transformed with the plasmids paagln, paacpsa and paacpsb were prepared as described in the Experimental Procedures and fractionated on a 1.5-ml Ni ++ -Probond column pre-equilibrated with 50 mm TrisHCl, ph 8, 10 mm 2-mercaptoethanol, and 200 mm NaCl. CPS.A (panel A), CPS.B (panel B) and GLN (panel C) were eluted with 200 mm imidazole in the same buffer and the column fractions were analyzed by SDS-polyacrylamide gel electrophoresis. 32

33 Figure 3. Gel permeation chromatography of the recombinant proteins. Panel A: The molecular mass of the A. aeolicus CPS.A (- -), CPS.B (- -) and a stoichiometric mixture of CPS.A and CPS.B (- -) was determined by chromatography on a Sephacryl S-300 HR column as described in the Experimental Procedures. The column was equilibrated and eluted with 50 mm potassium phosphate, ph 8, 10 mm 2-mercaptoethanol, and 200 mm NaCl. The ammonia-dependent CPSase activity (- -) in the fractions from CPS.A-CPS.B mixture was also assayed. Panel B: A stoichiometric mixture of GLN, CPS.A and CPS.B was also applied to the column and eluted under the conditions described in panel A. The absorbance (- -) and glutamine-dependent CPSase activity (- -) were measured and the fractions were analyzed by SDS-gel electrophoresis. The fractions on the gel are, from left to right, 79, 80, 82, 92, 97, 102, 107, 110, 113, 116, 117, 118, 119, 120, 123, 126, 129, 134. Figure 4. Thermostability CPS.A-CPS.B (- -) and GLN-CPS.A-CPS.B (- -) at a concentration of 6- and 1.4- mg/ml, respectively, in 50 mm potassium phosphate, ph 8, 10 mm 2-mercaptoethanol were incubated at the indicated temperatures for 10 min, quickly chilled on ice and assayed for ammonia-dependent CPSase (CPS.A-CPS.B) or glutamine-dependent CPSase (GLN-CPS.A- CPS.B) activities at 37 o C. Figure 5. Steady state kinetics of A. aeolicus CPSase Substrate saturation curves were measured at 78 o C by varying the concentration of one substrate and fixing the others at 30 mm ATP, 32 mm MgCl 2, 50 mm sodium bicarbonate, 200 mm NH 4 Cl or 2 mm glutamine in an assay buffer consisting of 6 mm aspartate, 3 µg of A. 33

34 aeolicus ATCase, 10 mm 2-mercaptoethanol, 0.05 M potassium phosphate, ph 8. Panel A: NH 4 Cl saturation curve for the ammonia-dependent CPSase activity of CPS.A-CPS.B (30 µg); Panel B: Bicarbonate saturation curve for the ammonia-dependent CPSase activity of CPS.A- CPS.B (30 µg); Panel C: ATP saturation curve for the ammonia-dependent CPSase activity of CPS.A-CPS.B (30 µg) with a 2 mm molar excess of MgCl 2 ; Panel D: NH 4 Cl (- -) and glutamine (- -) saturation curves of the CPSase activity of GLN-CPS.A-CPS.B (60 µg); Panel E: Bicarbonate saturation curve of ammonia-dependent (- -) and glutamine-dependent (- -) CPSase activity of GLN-CPS.A-CPS.B (200 µg); Panel F: ATP saturation curve for the ammonia-dependent (- -) and glutamine-dependent (- -) CPSase activity of GLN-CPS.A- CPS.B (60 µg) with a 2 mm molar excess of MgCl 2. Solid lines represent a non-linear least squares fit to the Michaelis-Menten or Hill equations (see legend to Table II). Lineweaver-Burke plots for each saturation curve are shown as inserts. The kinetic parameters are summarized in Table II. Figure 6. Allosteric Regulation of A. aeolicus CPSase The assays were conducted at 78 o C using the conditions described in the legend of Fig. 5, except that the ATP and MgCl 2 concentrations used in the experiments shown in Panels B and C were fixed at 5 mm and 7 mm, respectively. Panel A: ATP saturation curve of ammoniadependent CPSase activity of CPS.A-CPS.B (30 µg) in the absence of allosteric ligands (- -) or in the presence of 2 mm UMP (- -) or 5 mm ornithine (- -) and the glutamine-dependent CPSase activity of GLN-CPS.A-CPS.B (200 µg) in the absence of ligands (- -) or in the presence of 2 mm UMP (- -) or 5 mm ornithine (- -); Panel B: the ammonia-dependent 34

35 CPSase activity (- -) of CPS.A-CPS.B (90 µg) and the glutamine-dependent CPSase activity (- -) of GLN-CPS.A-CPS.B (200 µg) as a function of ornithine concentration; Panel C: the ammonia-dependent CPSase activity (- -) of CPS.A-CPS.B (90 µg) and the glutaminedependent CPSase activity (- -) of GLN-CPS.A-CPS.B (400 µg) as a function of UMP concentration. 35

36 Figure 1 Ahuja et al. A pyrb cara pyrc carb1 carb2 873 bp 1116 bp 276 Kb 1266 bp 267 Kb 1614 bp 1674 bp - 3 bp B A. aeolicus EFVAYTPYYYSSYERPYYTVDGQEILDE MSKKVVILGSGPNRIGQGIEFDYACVHA.: :..: :.. ::: :. E. coli EFATDTAYMYSTYEEECEA...NPSTDREKIMVLGGGPNRIGQGIEFDYCCVHA C CPS.A CPS.A CPS.B CPS.B Aaeo Eco Mjan Bsub Paer Hams CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B Aaeo Eco CPS.A CPS.B CPS.A CPS.B CPS.A CPS.B Mjan Bsub Paer CPS.A CPS.B Ham

37 Figure 2 Ahuja et al. A 64 kda Std B C column fractions 63 kda 45 kda Std column fractions 37