Characterization of the aspartate carbamoyltransferase fragment generated by protease action on the pyrimidine-3 gene product of Neurospora crassa.

The molecular weight of the fragment of aspartate carbamoyltransferase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) of Neurospora crassa following proteolysis was found to be 1.0-10(5) (aspartate carbamoyltransferase-L). It differs from the native form of the enzyme (aspartate carbamoyltransferase-N, 6.5-10(5)) in several respects. It has a lower V, has a much greater affinity (approx. 3-fold) for L-aspartate, and is strongly activated by glycine. Both forms of aspartate carbamyoltransferase have a pH optimum of approx. 9.5, and they exhibit similar affinities for carbamoyl phosphate.     

Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides.

Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide. Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.     

Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.     

Does ornithine stimulate carbamoylphosphate synthetase?

The effect of ornithine on carbamoylphosphate formation of rat liver mitochondria treated with Triton X 100 was studied. The rate of carbamoylphosphate accumulation and citrulline formation depended on the ATP-, Pi-, N-acetylglutamate and protein concentration. At optimal conditions the rate of citrulline formation was at least 1.5-fold higher than the rate at which carbamoylphosphate accumulated (ornithine absent). A significant correlation between the amount of carbamoylphosphate formed and the citrulline/carbamoylphosphate ratio (ornithine effect) was found. In mitochondria the presence of a carbamoylphosphate degrading enzyme could be demonstrated. This enzyme may be one factor for the differences in the rate of carbamoylphosphate accumulation and the rate of citrulline synthesis.     

Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: a case of by-product inhibition?

Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.     

Studies on the availability of intramitochondrial carbamoylphosphate for utilization in extramitochondrial reactions in rat liver

The following observations with isolated mitochondria prepared from rat liver demonstrate that Carbamoylphosphate can readily traverse the mitochondrial membrane: (a) Citrulline synthesis occurs within isolated intact mitochondria at the expense of exogenously added ornithine and [¹⁴C]carbamoylphosphate, providing evidence that the initochondrial membrane does not exclude extramitochondrial car bamoylphosphate from penetrating the intramitochondrial matrix, (b) The [¹⁴C]carbamoylphosphate synthesized within isolated intact mitochondria from NaH¹⁴CO₃ by the action of the N-acetyl-l-glutamate-activated carbamoylphosphate synthetase (CPS-I) is equally available for consumption in intramitochondrial and extramitochondrial reactions, as judged by the coupled activity of CPS-I with either intramitochondrial ornithine carbamoyltransferase or extramitochondrial aspartate carbamoyltransferase. The possibility that the coupled action of CPS-I with intramitochondrial ornithine carbamoyltransferase might prevent the export of carbamoylphosphate into the extramitochondrial medium was also examined. The addition of ornithine to the reaction mixture, at concentrations which are optimal for citrulline production, did not reduce carbamoylphosphate export below$\text{1}\text{3}$ of the total amount of carbamoylphosphate synthesized. These results indicate that the carbamoylphosphate generated intramitochondrially is not compartment ally excluded from participating in cytoplasmic reactions, and raise the possibility that the intramitochondrial carbamoylphosphate synthetase, CPS-I, may be a significant source of the carbamoylphosphate incorporated into hepatic pyrimidines by the cytoplasmic enzymes of the orotate pathway.     

Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.     

Regulation of mRNA levels of rat liver carbamoylphosphate synthetase by glucocorticosteroids and cyclic AMP as estimated with a specific cDNA

The construction and cloning of a cDNA complementary to the mRNA of rat liver carbamoylphosphate synthetase (ammonia) is described. Using this cDNA, the size of the mature, cytosolic carbamoylphosphate synthetase (ammonia) mRNA is estimated to be 6.0 Kb. The levels of carbamoylphosphate synthetase (ammonia) mRNA in liver are shown to be regulated by glucocorticosteroids and cyclic AMP. By studying mRNA levels of carbamoylphosphate synthetase, albumin and phosphoenolpyruvate carboxykinase, using specific cDNA clones, we show that carbamoylphosphate synthetase gene expression, like that of albumin is liver-specific.     

Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5'-phosphate: internal aldimine and stable L-aspartate external aldimine

The 1.8 ? resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5'-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and L-aspartate external aldimine forms are reported. The L-aspartate·deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with L-aspartate. This structure is compared to that of the α-methyl-L-aspartate·PLP external aldimine. Overlays with the corresponding pyridoxal 5'-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the L-aspartate·deazaPLP and α-methyl-L-aspartate·PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4'. In contrast, in the α-methyl-L-aspartate·PLP external aldimine, the ε-amino group of Lys258 is rotated ~70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.     

L-Aspartate-induced activation of aspartase

During the catalysis of the fumarate amination reaction, aspartase was markedly activated by the product, L-aspartate, as shown by a steep increase in the reaction rate. When NH4+ was replaced by NH2OH, the hydroxylamination reaction proceeded without any acceleration, and was activated upon addition of L-aspartate. The activation required the Mg2+ ion and the alkaline pH, and the half-saturation concentration of L-aspartate for activation was as low as 0.07 mM, which was far lower than the Km value for catalysis. Fumarate showed no activating effect in contrast to L-aspartate, and L-aspartate lowered the Km value for fumarate instead of acting as a competitive inhibitor. Besides L-aspartate, alpha-methyl-DL-aspartate exhibited an activating effect without serving as a substrate. These results suggest that the activation is mediated by an indirect action of L-aspartate which is bound to a site distinct from the catalytic site.     

13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase.

13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme. The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor). The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP. The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same. Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group. Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate. An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate.     

Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.     

Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius. Cloning, sequence analysis, enzyme purification and characterization.

The genes coding for aspartate carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6-50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of L-aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.     

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.     

Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.     

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.     

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.