Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases.

Carbamoyl phosphate synthetases (CPSs) utilize either glutamine or ammonia for the ATP-dependent generation of carbamoyl phosphate. In glutamine-utilizing CPSs (e.g. the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at a triad-type glutamine amidotransferase domain. Non-glutamine-utilizing CPSs (e.g. rat and human CPS I), lacking the catalytic cysteine residue, can generate carbamoyl phosphate only in the presence of free ammonia. Frog CPS I (fCPS I), unlike mammalian CPS Is, retains most of the glutamine amidotransferase residues conserved in glutamine-utilizing CPSs, including an intact catalytic triad, and could therefore be expected to use glutamine. Our work with native fCPS I provides the first demonstration of the inability of this enzyme to bind/utilize glutamine. To determine why fCPS I is unable to utilize glutamine, we compared sequences of glutamine-using and non-glutamine-using CPSs to identify residues that are present or conservatively substituted in all glutamine-utilizing CPSs but absent in fCPS I. We constructed the site-directed mutants Q273E, L270K, Q273E/N240S, and Q273E/L270K in E. coli CPS and have determined that simultaneous occurrence of the two substitutions, Gln-->Glu and Leu-->Lys, found in the frog CPS I glutamine amidotransferase domain are sufficient to eliminate glutamine utilization by the E. coli enzyme.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Role of the hinge loop linking the N- and C-terminal domains of the amidotransferase subunit of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit and a small amidotransferase subunit. The small subunit is structurally bilobal. The N-terminal domain is unique compared to the sequences of other known proteins. The C-terminal domain, which contains the direct catalytic residues for the amidotransferase activity of CPS, is homologous to other members of the Triad glutamine amidotransferases. The two domains are linked by a hinge-like loop, which contains a type II beta turn. The role of this loop in the hydrolysis of glutamine and the formation of carbamoyl phosphate was probed by site-directed mutagenesis. Based upon the observed kinetic properties of the mutants, the modifications to the small subunit can be separated into two groups. The first group consists of G152I, G155I, and Delta155. Attempts to disrupt the turn conformation were made by the deletion of Gly-155 or substitution of the two glycine residues with isoleucine. However, these mutations only have minor effects on the kinetic properties of the enzyme. The second group includes L153W, L153G/N154G, and a ternary complex consisting of the intact large subunit plus the separate N- and C-terminal domains of the small subunit. Although the ability to synthesize carbamoyl phosphate is retained in these enzymes, the hydrolysis of glutamine is partially uncoupled from the synthetase reaction. It is concluded that the hinge loop, but not the type-II turn structure of the loop per se, is important for maintaining the proper interface interactions between the two subunits and the catalytic coupling of the partial reactions occurring within the separate subunits of CPS.     

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Human carbamoyl phosphate synthetase (hCPS) has evolved critical features that allow it to remove excess and potentially neurotoxic ammonia via the urea cycle, including use of only free ammonia as a nitrogen donor, a K_m for ammonia 100-fold lower than for CPSs that also use glutamine as a nitrogen donor, and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. The recent availability of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches to elucidate the distinctive hCPS properties. Although the site of AGA interaction is not defined, it is known that the binding of AGA to CPS leads to a conformational change in which a pair of cysteine side chains become proximate and can then be selectively induced to undergo disulfide bonding. We analyzed the response of hCPS cysteine mutants to thiol-specific reagents and identified Cys-1327 and Cys-1337 as the AGA-responsive proximate cysteines. Possibly two of the features unique to urea-specific CPSs, relative to other CPSs (the conserved Cys-1327/Cys-1337 pair and the occurrence at very high concentrations in the liver mitochondrial matrix) co-evolved to provide buffering against reactive oxygen species. Reciprocal mutation analysis of Escherichia coli CPS (eCPS), creating P909C and G919C and establishing the ability of these engineered cysteine residues to share a disulfide bond, indicated an eCPS conformational change at least partly similar to the hCPS conformational change induced by AGA. These findings strongly suggested an alternative eCPS conformation relative to the single crystal conformation thus far identified.When life adapted to a terrestrial habitat, removal of excess and potentially neurotoxic ammonia by the diffusion that occurred in an aquatic habitat was no longer possible (1). Arginine biosynthetic pathways almost certainly served as the precursors for the urea cycle, the present day metabolic pathway for removal of excess ammonia, with surprisingly few changes needed for the pathway evolution (2). Carbamoyl phosphate synthetase (CPS),² the enzyme that catalyzes the entry and rate-limiting step of the urea cycle, was the site of four of these critical evolutionary changes: (a) gain of communication with a sensor of excess amino acids, N-acetylglutamate (AGA, which serves as a required allosteric activator only for urea-synthesizing CPSs), (b) a decrease in K_m for ammonia to ∼1 mm from the ∼100 mm value for other CPSs, (c) loss of interaction with glutamine to avoid competition with the preferred substrate ammonia, and (d) localization to the hepatic mitochondrial matrix to allow independent regulation relative to CPSs with other metabolic roles. Structural changes have been identified in hCPS that correlate with the latter two functional changes (3-5); the cysteine residue required for releasing ammonia from glutamine has been replaced by serine, and hCPS is synthesized with 39 N-terminal residues that serve as a mitochondrial matrix targeting signal and that are cleaved as the hCPS precursor crosses the inner mitochondrial membrane. The structural basis for the first two critical functional features, AGA interaction and high affinity for ammonia, however, have not yet been elucidated nor has it been determined whether these two features are linked.CPSs from all species with varied metabolic roles share strong sequence identity, and all appear to have the same overall domain organization (6) (Fig. 1) observed in the crystal structure of E. coli CPS (eCPS), the only solved CPS structure (7). All known CPSs appear to utilize a common mechanism (Fig. 1) to catalyze the formation of carbamoyl phosphate (CP), P_i, and two molecules of ADP from ammonia (either free in the cell or generated from the hydrolysis of glutamine), bicarbonate, and two molecules of ATP (6). A variety of studies have yielded evidence for a cycle of conformational changes accompanying the catalytic cycle (6-8). Vertebrate urea-specific CPSs, including hCPS, incorporate an additional type of conformational control in the form of dependence on the allosteric activator AGA. In the absence of AGA, a very small fraction of urea-specific CPS is in a conformation capable of catalytic activity, whereas binding of AGA yields CPS with activity equivalent to that of eCPS and other non-AGA-dependent CPSs (6). Although AGA functions as an intermediate in arginine biosynthesis in prokaryotes and lower eukaryotes, the only known function for AGA in vertebrates is activation of urea-specific CPSs (9). Co-localization of AGA synthetase in the hepatic mitochondrial matrix is consistent with this dedicated role, as is the fact that increases in the substrate glutamate signal increased degradation of proteins to yield both free amino acids and free ammonia. Allosteric activation of vertebrate AGA synthetase by arginine provides a further link to the presence of excess amino acids and the accompanying excess of free ammonia requiring detoxification.Open in a separate windowFIGURE 1.Domain and oligomeric structure of eCPS. Top panel, ribbon representation of one heterodimer of eCPS. The 42-kDa glutaminase subunit contains the glutamine binding domain (A2; violet) and domain A1 (purple) that is involved in communicating active site occupancy between the glutaminase and synthetase subunits. The 120-kDa synthetase subunit contains 4 domains. Domains B (green) and C (red) are regions of internal duplication, and each contains an ATP grasp fold. The oligomerization domain (D′, yellow) and allosteric domain (D, orange) are involved in side-by-side and end-on-end interactions, respectively. The ligands glutamine and ADP are shown in space-fill representations. The intramolecular tunnel connecting the three active sites is illustrated with arrows. Also shown are the individual chemical reactions at each of the active sites that are synchronized in the overall reaction. The domains of hCPS occur as a single polypeptide, with 1462 amino acid residues in the mature protein (from which the mitochondrial targeting signal has been removed) in contrast to the heterodimeric structure of eCPS. Bottom panel, the tetramer of the glutaminase + synthetase heterodimers. Created from PDB file 1a9x.One reporter for the AGA-activated conformation of CPS is a pair of cysteine side chains that become proximate upon AGA binding and that can then be selectively induced to undergo disulfide bonding (10, 11). In the absence of AGA, only a small fraction of CPS is in a conformation with the cysteine pair proximate. This single pair of proximate sulfhydryl groups is uniquely modified when the AGA·CPS complex is exposed to a variety of disulfide-inducing reagents, and reversible activity loss accompanies disulfide formation. The recent availability (12) of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches for elucidation of the distinctive hCPS properties. To identify the residues of the cysteine reporter group, we have analyzed the response of hCPS cysteine mutants to thiol-specific reagents. We also have examined the potential for a conformational change in eCPS that parallels the AGA-induced conformational change of hCPS. Thus far only a single conformation of eCPS has been revealed from x-ray structural analysis. Defining the cycle of conformational changes that accompany the catalytic cycle and that allow synchronization of three active sites (glutamine amidotransferase site and two distinct ATP sites) to produce CP, therefore, remains a major challenge.     

Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase

Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.     

Perforation of the tunnel wall in carbamoyl phosphate synthetase derails the passage of ammonia between sequential active sites

Carbamoyl phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (approximately 42 kDa) and a large subunit (approximately 118 kDa) and catalyzes the biosynthesis of carbamoyl phosphate from MgATP, bicarbonate, and glutamine. The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent. CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 A. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation. Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall. A hole in the tunnel wall was made by mutation of integral amino acid residues with alanine residues. The triple mutant alphaP360A/alphaH361A/betaR265A was unable to utilize glutamine for the synthesis of carbamoyl phosphate. However, the mutant enzyme retained full catalytic activity when external ammonia was used as the nitrogen source. The synchronization of the partial reactions occurring at the three active sites observed with the wild-type CPS was seriously disrupted with the mutant enzyme when glutamine was used as a nitrogen source. Overall, the catalytic constants of the mutant were consistent with the model where the channeling of ammonia has been disrupted due to the leakage from the ammonia tunnel to the protein exterior.     

The differentially conserved residues of carbamoyl-phosphate synthetase

Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein. The larger of the two subunits (M(r) approximately 118,000) contains a pair of homologous domains of approximately 400 residues each that are approximately 40% identical in amino acid sequence. The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain approximately 79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other. The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled. True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other. Neither of the two possible chimeric proteins was structurally stable. These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent. This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues.     

Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

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.     

Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.     

Localization of the site for the nucleotide effectors of Escherichia coli carbamoyl phosphate synthetase using site-directed mutagenesis

Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP. The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished. The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere. Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP.     

The small subunit of carbamoyl phosphate synthetase: snapshots along the reaction pathway

Carbamoyl phosphate synthetase (CPS) plays a key role in both arginine and pyrimidine biosynthesis by catalyzing the production of carbamoyl phosphate. The enzyme from Escherichi coli consists of two polypeptide chains referred to as the small and large subunits. On the basis of both amino acid sequence analyses and X-ray structural studies, it is known that the small subunit belongs to the Triad or Type I class of amidotransferases, all of which contain a cysteine-histidine (Cys269 and His353) couple required for activity. The hydrolysis of glutamine by the small subunit has been proposed to occur via two tetrahedral intermediates and a glutamyl-thioester moiety. Here, we describe the three-dimensional structures of the C269S/glutamine and CPS/glutamate gamma-semialdehyde complexes, which serve as mimics for the Michaelis complex and the tetrahedral intermediates, respectively. In conjunction with the previously solved glutamyl-thioester intermediate complex, the stereochemical course of glutamine hydrolysis in CPS has been outlined. Specifically, attack by the thiolate of Cys269 occurs at the Si face of the carboxamide group of the glutamine substrate leading to a tetrahedral intermediate with an S-configuration. Both the backbone amide groups of Gly241 and Leu270, and O(gamma) of Ser47 play key roles in stabilizing the developing oxyanion. Collapse of the tetrahedral intermediate leads to formation of the glutamyl-thioester intermediate, which is subsequently attacked at the Si face by an activated water molecule positioned near His353. The results described here serve as a paradigm for other members of the Triad class of amidotranferases.     

The C-terminal domain of HPII catalase is a member of the type I glutamine amidotransferase superfamily

Discovering distant evolutionary relationships between proteins requires detecting subtle similarities. Here we use a combination of sequence and structure analysis to show that the C-terminal domain of Escherichia coli HPII catalase with available spatial structure is a divergent member of the type I glutamine amidotransferase (GAT) superfamily. GAT-containing proteins include many biosynthetic enzymes such as E. coli carbamoyl phosphate synthetase and anthranilate synthase. Typical GAT domains have Rossmann fold-like topology and possess a catalytic triad similar to that of proteases. The C-terminal domain of HPII catalase has the GAT Rossmann fold but lacks the triad and therefore loses enzymatic activity. In addition, we detect significant sequence similarity between thiJ domains, some of which are known to have protease activity, and typical GAT proteins. Evolutionary tree analysis of the entire GAT superfamily indicates that the HPII catalase is more closely related to thiJ domains than to classical GAT domains and is likely to have evolved from a thiJ-like protein. This work illustrates the strength of sequence-based profile analysis techniques coupled with structural superpositions in developing an evolutionarily relevant classification of protein structures. Proteins 2001;42:230-236.     

Deconstruction of the catalytic array within the amidotransferase subunit of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit, and a small amidotransferase subunit, which belongs to the Triad family of glutamine amidotransferases. Previous studies have established that the reaction mechanism of the small subunit proceeds through the formation of a gamma-glutamyl thioester with Cys-269. The roles in the hydrolysis of glutamine played by the conserved residues, Glu-355, Ser-47, Lys-202, and Gln-273, were determined by mutagenesis. In the X-ray crystal structure of the H353N mutant, Ser-47 and Gln-273 interact with the gamma-glutamyl thioester intermediate [Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, 8825-8831]. The mutants E355D and E355A have elevated values of K(m) for glutamine, but the overall carbamoyl phosphate synthesis reaction is unperturbed. E355Q does not significantly affect the bicarbonate-dependent ATPase or glutaminase partial reactions. However, this mutation almost completely uncouples the two partial reactions such that no carbamoyl phosphate is produced. The partial recovery of carbamoyl phosphate synthesis activity in the double mutant E355Q/K202M argues that the loss of activity in E355Q is at least partly due to additional interactions between Gln-355 and Lys-202 in E355Q. The mutants S47A and Q273A have elevated K(m) values for glutamine while the V(max) values are comparable to that of the wild-type enzyme. It is concluded that contrary to the original proposal for the catalytic triad, Glu-355 is not an essential residue for catalysis. The results are consistent with Ser-47 and Gln-273 playing significant roles in the binding of glutamine.     

Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme. These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites. The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification. The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a substantially reduced rate of carbamoyl phosphate synthesis, but the rate of ATP turnover and glutamine hydrolysis was not significantly altered. These data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain. The partial reactions of CPS have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active sites has not been functionally perturbed.     

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

Nucleotide recognition in the ATP-grasp protein carbamoyl phosphate synthetase

Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle on duplicated nucleotide-binding sites (N and C). To clarify the contributions of sites N and C to the overall reaction, we carried out site-directed mutagenesis aimed at changing the substrate specificity of either of the two sites from ATP to GTP. Mutant design was based in part on an analysis of the nucleotide-binding sites of succinyl-CoA synthetases, which share membership in the ATP-grasp family with CPS and occur as GTP- and ATP-specific isoforms. We constructed and analyzed Escherichia coli CPS single mutations A144Q, D207A, D207N, S209A, I211S, P690Q, D753A, D753N, and F755A, as well as combinations thereof. All of the mutants retained ATP specificity, arguing for a lack of plasticity of the ATP sites of CPS with respect to nucleotide recognition. GTP-specific ATP-grasp proteins appear to accommodate this substrate by a displacement of the base relative to the ATP-bound state, an interaction that is precluded by the architecture of the potassium-binding loop in CPS. Analysis of the ATP-dependent kinetic parameters revealed that mutation of several residues conserved in ATP-grasp proteins and CPSs had surprisingly small effects, whereas constructs containing either A144Q or P690Q exerted the strongest effects on ATP utilization. We propose that these mutations affect proper movement of the lids covering the active sites of CPS, and interfere with access of substrate.     

The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain.     

A novel carbamoyl-phosphate synthetase from Aquifex aeolicus

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 glutaminase, CPS.A, and CPS.B homologs from A. aeolicus were cloned, overexpressed in Escherichia 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 glutaminase 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 of the residues known to be essential for function are conserved and properly positioned. A steady state kinetic study at 78 degrees C indicated that although 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.     

A functional analysis of the allosteric nucleotide monophosphate binding site of carbamoyl phosphate synthetase

The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.