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.     

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.     

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.     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

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.     

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.     

Resolving the fluorescence response of Escherichia coli carbamoyl phosphate synthetase: mapping intra- and intersubunit conformational changes

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induces fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis.     

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.     

Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.     

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.     

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.     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.     

Suicide inactivation of fructose-1,6-bisphosphate aldolase

2-Keto-4,4,4-trifluorobutyl phosphate (HTFP) was prepared from 3,3,3-trifluoropropionic acid. HTFP acts as an irreversible inhibitor of rabbit muscle aldolase: the loss of activity was time dependent and the inactivation followed a pseudo-first-order process. Values of 1.4 mM for the dissociation constant and 2.3 X 10(-2) s-1 for the reaction rate constant were determined. The kinetic constants do not depend on the enzyme concentration. No effect of thiols on the inactivation rate was detected. Only 1-2 mol of fluoride ions was liberated per inactivated subunit, indicative of a low partition ratio. Dihydroxyacetone phosphate protected the enzyme against the inactivation in a competitive manner, and glyceraldehyde 3-phosphate protected as if it formed a condensation product with HTPF. 5,5'-Dithiobis(2-nitrobenzoic acid) thiol titration showed the loss of one very reactive thiol group per enzyme subunit after inactivation. All those observations seem to agree with a suicide substrate inactivation of aldolase by HTPF.     

Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.     

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.     

Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes

Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate from glutamine or ammonia, bicarbonate and ATP. There are three different isoforms of CPS that play vital roles in two metabolic pathways, pyrimidine biosynthesis (CPS II) and arginine/urea biosynthesis (CPS I and CPS III). Gene duplication has been proposed as the evolutionary mechanism creating this gene family with CPS II likely giving rise to the CPS I/III clade. In the evolutionary history of this gene family it is still undetermined when CPS I diverged from CPS III on the path to terrestriality in the vertebrates. Transitional organisms such as lungfishes are of particular interest because they are capable of respiring via gills and with lungs and therefore can be found in both aquatic and terrestrial environments. Notably, enzymatic characterization of the mitochondrial CPS isoforms in this transitional group has not led to clear conclusions. In order to determine which CPS isoform is present in transitional animals, we examined partial sequences for liver CPS amplified from five species of lungfish, and a larger fragment of CPS from one lungfish species (Protopterus annectens) and compared them to CPS isoforms from other fish and mammals. Enzyme activities for P. annectens liver were also examined. While enzyme activities did not yield a clear distinction between isoforms (virtually equal activities were obtained for either CPS I or III), CPS sequences from the lungfishes formed a monophyletic clade within the CPS I clade and separate from the CPS III clade of other vertebrates. This finding implies that the mitochondrial isoform of CPS in lungfish is derived from CPS I and is likely to have a physiological function similar to CPS I. This finding is important because it supports the hypothesis that lungfish employ a urea cycle similar to terrestrial air-breathing vertebrates.     

The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate.

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

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.