An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

Long-range allosteric transitions in carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.     

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.     

Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.     

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.     

Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations

Carbamoyl phosphate synthetase (CPS) is a member of the amidotransferase family of enzymes that uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations. Molecular dynamics simulations for the transfer of ammonia and ammonium through a tunnel in the small subunit of CPS resulted in five successful trajectories for ammonia transfer, while ammonium was immobilized in a water pocket inside the small subunit of the heterodimeric protein. The observed molecular tunnel for ammonia transport is consistent with that suggested by earlier X-ray crystallography and site-directed mutation studies. His-353, Ser-47, and Lys-202, around the active site center in the small subunit, function cooperatively to deliver ammonia from the site of formation to the interface with the large subunit, via the exchange of hydrogen bonds with a critical water cluster within the tunnel. The NH 3 forms and breaks hydrogen bonds to Gly-292, Ser-35, Pro-358, Gly-293, and Thr-37 in a stepwise fashion "macroscopically" as it travels through the hydrophilic passage toward the subunit interface. The potential of mean force calculations along the ammonia transfer pathway indicates a low free-energy path for the translocation of ammonia with two barriers of 3.9 and 5.5 kcal/mol, respectively. These low free-energy barriers are consistent with the delivery of ammonia from the site of formation into a water reservoir toward the exit of the tunnel and migration through the hydrophilic leaving passage, respectively. The high overall free-energy barrier of 22.4 kcal/mol for the transport of ammonium additionally substantiates that the tunnel in the small subunit of CPS is not an ammonium but an ammonia channel.     

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.     

Access to the carbamate tunnel of carbamoyl phosphate synthetase

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli revealed the existence of a molecular tunnel that has been proposed to facilitate the translocation of reaction intermediates between remotely located active sites. Five highly conserved glutamate residues, including Glu-25, Glu-383, Glu-577, Glu-604, and Glu-916, are close together in two clusters in the interior wall of the molecular tunnel that enables the intermediate carbamate to migrate from the site of synthesis to the site of utilization. Two arginines, Arg-306 and Arg-848, are located at either end of the carbamate tunnel and participate in the binding of ATP at each of the two active sites within the large subunit of CPS. The mutation of Glu-25 or Glu-577 results in a diminution in the overall rate of carbamoyl phosphate formation. Similar effects are observed upon mutation of Arg-306 and Arg-848 to alanine residues. The conserved glutamate and arginine residues may function in concert with one another to control entry of carbamate into the tunnel prior to phosphorylation to carbamoyl phosphate. The electrostatic environment of tunnel interior may help to stabilize the tunnel architecture and prevent decomposition of carbamate through protonation.     

Conversion of carbamoyl phosphate to hydroxyurea. An assay for carbamoylphosphate synthetase

A specific colorimetric method for determination of hydroxyurea is described. Using this method it was shown that carbamoyl phosphate or cyanate (a decomposition product of carbamoyl phosphate) and hydroxylamine react to form hydroxyurea. Hydroxylamine was employed to trap carbamoyl phosphate produced by carbamoylphosphate synthetase. A radio chemical assay for activity of carbamoylphosphate synthetase gives results in agreement with the method based on coupling the formation of carbamoyl phosphate with ornithine transcarbamoylase. This new assay is sensitive, rapid, and reproducible, and does not require supplementary enzymes or their substrates in the incubation medium.     

Synchronization of the three reaction centers within carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from E. coli catalyzes the synthesis of carbamoyl phosphate through a series of four reactions occurring at three active sites connected by a molecular tunnel of 100 A. To understand the mechanism for coordination and synchronization among the active sites, the pre-steady-state time courses for the formation of phosphate, ADP, glutamate, and carbamoyl phosphate were determined. When bicarbonate and ATP were rapidly mixed with CPS, a stoichiometric burst of acid-labile phosphate and ADP was observed with a formation rate constant of 1100 min(-)(1). The burst phase was followed by a linear steady-state phase with a rate constant of 12 min(-)(1). When glutamine or ammonia was added to the initial reaction mixture, the magnitude and the rate of formation of the burst phase for either phosphate or ADP were unchanged, but the rate constant for the linear steady-state phase increased to an average value of 78 min(-)(1). These results demonstrate that the initial phosphorylation of bicarbonate is independent of the binding or hydrolysis of glutamine. The pre-steady-state time course for the hydrolysis of glutamine in the absence of ATP exhibited a burst of glutamate formation with a rate constant of 4 min(-)(1) when the reaction was quenched with base. In the presence of ATP and bicarbonate, the rate constant for the formation of the burst of glutamate was 1100 min(-)(1). The hydrolysis of ATP thus enhanced the hydrolysis of glutamine by a factor of 275, but there was no effect by glutamine on the initial phosphorylation of bicarbonate. The pre-steady-state time course for the formation of carbamoyl phosphate was linear with an overall rate constant of 72 min(-)(1). The absence of an initial burst of carbamoyl phosphate formation eliminates product release as a rate-determining step for CPS. Overall, these results have been interpreted to be consistent with a mechanism whereby the phosphorylation of bicarbonate serves as the initial trigger for the rest of the reaction cascade. The formation of the carboxy phosphate intermediate within the large subunit must induce a conformational change to the active site of the small subunit that enhances the hydrolysis of glutamine. Thus, ammonia is not released into the molecular tunnel until the activated bicarbonate is ready to form carbamate. The rate-limiting step for the steady-state assembly of carbamoyl phosphate is either the formation, migration, or phosphorylation of the carbamate intermediate.     

Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.     

Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase.

The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.     

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.     

Control of ureogenesis

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made. Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium. The relationship between ornithine-cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near-physiological conditions the ornithine-cycle enzymes are far from being saturated with their subsidiaries. Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine. Activation of carbamoyl-phosphate synthetase (ammonia) by addition of N-carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine-cycle flux to the steady-state ammonia concentration was shifted to lower concentrations of ammonia. The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl-phosphate synthetase. It is concluded that in vivo the function of activity changes in carbamoyl-phosphate synthetase, via the well-documented alterations in the intramitochondrial concentration of N-acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl-phosphate synthetase.     

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.     

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.     

Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain.

The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site.     

Aquifex aeolicus aspartate transcarbamoylase, an enzyme specialized for the efficient utilization of unstable carbamoyl phosphate at elevated temperature

Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.     

Urea-hydrolysis-dependent citrulline synthesis by Ureaplasma urealyticum

Some of the ammonia produced by hydrolysis of urea by Ureaplasma urealyticum is channelled into an anabolic pathway with resultant 'de novo' synthesis of citrulline. The organism appears to possess ornithine carbamoyltransferase and carbamoyl phosphate synthetase or some modified form of these enzymes.