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.     

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.     

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.     

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.     

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 apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ.

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.     

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.     

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.     

Activation of carbamoyl phosphate synthetase by cryoprotectants

Molecular and Cellular Biochemistry - Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryoprotectants. Their diverse chemical structure and the normal...     

Carbamoyl phosphate synthetase: a tunnel runs through it

The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 Å in length was observed. The recently determined three-dimensional structure of carbamoyl phosphate synthetase sets a new long distance record in that the three active sites are separated by nearly 100 Å.     

Characterization of carbamoyl phosphate synthetase of Streptomyces spp

Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).     

Kinetic studies of bovine liver carbamoyl phosphate synthetase

A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO₃⁻, ATPMg and NH₄⁺. A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Domain structure of rat liver carbamoyl phosphate synthetase I

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.     

Some regulatory properties of pea leaf carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase of pea shoots (Pisum sativum L.) was purified 101-fold. Its stability was greatly increased by the addition of substrates and activators. The enzyme was strongly inhibited by micromolar amounts of UMP (Ki less than 2 mum). UDP, UTP, TMP, and ADP were also inhibitory. AMP caused either slight activation (under certain conditions) or was inhibitory. Uridine nucleotides were competitive inhibitors, as was AMP, while ADP was a noncompetitive inhibitor. Enzyme activity was increased manyfold by the activator ornithine. Ornithine acted by increasing the affinity for Mg.ATP by a factor of 8 or more. Other activators were IMP, GMP, ITP, and GTP, IMP, like ornithine, increased the Michaelis constant for Mg.ATP. The activators ornithine, GMP, and IMP (but not GTP and ITP) completely reversed inhibition caused by pyrimidine nucleotides while increasing the inhibition caused by ADP and AMP.     

Product inhibition studies on bovine liver carbamoyl phosphate synthetase

A study of the product-inhibition patterns of carbamoyl phosphate synthetase from bovine liver is reported. Inhibition by adenosine, AMP and inorganic ions is also reported. The results are in agreement with the previously proposed model in which the order of substrate binding is ATPMg, followed by HCO(3) (-), ATPMg and NH(4) (+). The order of product release on the basis of the reported results is carbamoyl phosphate, followed by ADPMg, ADPMg and inorganic phosphate.     

Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group

The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.     

Immunoferritin location of carbamoyl phosphate synthetase in rat liver.

Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.     

Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.     

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.     

Schistosoma mansoni: suppression of carbamoyl phosphate synthetase (ammonia) and ornithine carbamoyltransferase activities in the liver of infected mice

ICR female mice infected with cercariae of Schistosoma mansoni exhibited a significant decrease in both total and specific activities of carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine carbamoyltransferase (EC 2.1.3.3), and also in the serum urea level. Intraperitoneal administration of the S. mansoni egg granulomas or 15,000g X 30 min supernatant fluid of their extract into the uninfected, normal mice also significantly decreased the total and specific activities of both enzymes without any appreciable histopathological influence on their livers. S. mansoni viable eggs caused a significant decrease in the total and specific activities of carbamoyl phosphate synthetase (ammonia) alone as well as active intraperitoneal inflammation when inoculated into the normal mice by the same route. There was no difference in the amount of food intake between the control and these experimental mice. These findings suggest that the granuloma or inflammatory cells induced by schistosome eggs produce some factor(s) which may be responsible for reduction of these enzymatic activities in experimental schistosomiasis mansoni.