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Using UV-irradiation we cross-linked IMP, the allosteric activator of E. coli carbamyl phosphate synthetase (a heterodimer of 117.7 and 41.4 kDa subunits), to the large subunit of the enzyme. As in the native enzyme-IMP complex, the cross-linked complex was resistant to attack by trypsin. Thus, IMP is attached to its normal site and induces the normal conformational changes. Limited digestion of the [3H]IMP-labeled enzyme with V8 staphylococcal protease or with trypsin in the presence of SDS, and NH2-terminal sequencing, showed that [3H]IMP is cross-linked to the COOH-terminal 20 kDa domain of the large subunit, downstream of residue 912, supporting the proposal that this domain is specialized in effector binding and regulation.

The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.

The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low-affinity T state to a high-affinity R state. In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate. It is shown here that cooperativity for this substrate is only apparent. Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state. Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T-->R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate. Similar results were obtained in the course of continuous-flow-dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state. Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.

A continuous spectrophotometric method for assaying ATCase activity has been devised that couples the production of inorganic phosphate from the ATCase-catalyzed reaction to the phosphorolysis reaction catalyzed by purine nucleoside phosphorylase, using a chromophoric nucleotide analogue, methylthioguanosine (MESG). This latter reaction results in a change in extinction coefficient of 11,000 M-1 cm-1 at 360 nm, providing a means for continuous assay of ATCase activity by spectrophotometry in the visible light region. This delta epsilon 360 is sufficiently large to allow continuous determination of reaction rates with micromolar levels of carbamyl-phosphate, a feature not offered by other currently used assay methods. Other currently available ATCase assay methods typically include fixed-time incubations involving [14C]Asp that require multiple chromatographic separations, colorimetry requiring long incubations with corrosive chemicals in the dark, or relatively insensitive continuous approaches involving a pH stat or far uv spectrophotometry. This facile, inexpensive MESG-coupled assay can be routinely applied to studies of ATCase altered by feedback modifiers or by site-specific mutations. Saturation curves for Asp and CP determined by other methods at pH 7 and 8 have been reproduced by the MESG/PNP-coupled approach. The kinetic binding of CP was demonstrated to be non-cooperative at low [Asp], i.e., under conditions at which ATCase was primarily in the T state. Cooperative binding of CP observed under conditions of saturating [Asp] (i.e., with ATCase in the R state) appears to reflect binding of Asp rather than CP.(ABSTRACT TRUNCATED AT 250 WORDS)

Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction. In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline. Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate. Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g. spermidine). A previous study showed that a modification of the C-terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme. We have now investigated the importance of the C-terminus for homotropic and heterotropic cooperativity by site-directed mutagenesis. Deletion of the C-terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine. These properties were essentially restored when the two C-terminal amino acids (Asp334 and Ile335) were removed by deletion. However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild-type enzyme was rendered virtually non-cooperative by AMP. An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity. All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine. A structural model of the dodecamer, at 0.3-nm resolution, suggests that the C-terminus could be involved in trimer/trimer interaction. We propose that modifications at the C-terminus alter the trimer/trimer interface and, in addition, removes the salt bridge His5-Ile335 within a monomer. These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors.

Ornithine transcarbamylase (OTCase) has been purified from porcine liver by a simple four-step procedure that included chromatography on an affinity column to which the transition-state analogue, delta-N-phosphonacetyl-L-ornithine (PALO), was covalently bound. The procedures employed yielded an enzyme which was purified some 260-fold and was judged to be homogeneous by nondenaturing- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent homogeneity of the enzyme was confirmed by N-terminal sequence analysis. The molecular weight of the porcine enzyme was determined by Sephadex gel exclusion chromatography and sedimentation equilibrium. An approximate molecular weight of 107,000 was calculated by both procedures. The single band obtained by SDS-PAGE indicated a subunit molecular weight of 36,800 +/- 700; hence, the enzyme is a trimer of identical subunits. The sedimentation coefficient of the native enzyme was determined to be 6.47. At pH 8.0, the Km values for the substrates are 0.41 and 1.3 mM for ornithine and carbamyl phosphate, respectively. PALO is a competitive inhibitor and has a Ki of 0.13 microM, which suggests that it binds with about 10,000 times greater affinity than carbamyl phosphate. Amino acid analysis performed on acid hydrolyzed enzyme yielded 323 amino acids per monomer. Performic acid oxidation of the enzyme, followed by acid hydrolysis and amino acid analysis, showed three cysteine residues per subunit. A partial specific volume of 0.725 cc/g was calculated from the amino acid composition. Reaction of purified porcine OTCase with phenylglyoxal, an arginine-specific reagent, results in complete loss of catalytic activity. The decrease in enzymatic activity correlates with the modification of 1 mol of arginine per mole of OTCase monomer. In the presence of 20 mM carbamyl phosphate, 93% of the activity is retained during a 1-h reaction time. Other substrates and substrate combinations offer less protection.

The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.

The proximal promoter of the rat carbamyl phosphate synthetase I gene contains four cis-acting regulatory regions, designated sites GAG, I, II, and III. The GAG site, which is located adjacent to the predicted TATA region, contains a direct repeat of the nonamer, GAGGAGGGG, and binds a factor which is unrelated to the other upstream binding site factors. Sites I-III, on the other hand, bind the same or similar factors, but with different affinities (site II > site III > site I). High affinity binding to site II requires the core octamer, GTTGCAAC. Sequential 5'-deletion of the promoter revealed that GAG is the predominant activating element in transient transfection analyses and, on its own, can sustain activity of a -67 promoter fragment. However, in the context of a -157 promoter in which the GTTGCAAC core of site II had been mutated, promoter activity was completely repressed, and this repression was due to sites I and III. Repression by sites I and III in the presence of mutated site II was relieved either by mutating sites I and III or by introducing a 76-bp random DNA fragment between GAG and site I. Our results suggest a model in which the carbamyl phosphate synthetase I promoter is controlled by a single activating element, GAG, and by two repressor elements, sites I and III. We conclude that the high affinity site II element binds an anti-repressor. Footprint analyses of the -157 promoter with and without a mutation of the GTTGCAAC element in site II suggest that the anti-repressor may interfere with the site I and site III repressors by quenching.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GOT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

Inactivations of chicken liver pyruvate carboxylase with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) and o-phthalaldehyde (o-PA) have identified cysteine and lysine residues that are essential for catalytic activity. Protection experiments suggest that the modified residues are located in or near the first and second subsites. At a one- to two-fold molar excess over active site concentration, DACM inactivated approximately 80-90% of the pyruvate carboxylase and ADP/Pi linked oxaloacetate decarboxylase activities by forming a sulfhydryl-DACM adduct with a fluorescence excitation maximum at 385 nm and an emission maximum at 476 nm. o-PA reacted with the enzyme by cross-linking lysine and cysteine residues to form an inactive isoindole-enzyme derivative with a fluorescence excitation maximum at 337 nm and an emission maximum at 415 nm. Incorporation of one equivalent of either DACM or isoindole derivative resulted in an 80-90% decrease in all activities involving chemistry at the first subsite, suggesting that the modification of a sulfhydryl group or a cysteine-lysine ion pair in or near the first subsite inactivates the enzyme. A cysteine-lysine ion pair in the first subsite could function to remove the N-1 proton of biotin to yield enol-biotin, which could be readily carboxylated by the carboxyphosphate intermediate. In the reverse direction, a cysteine-lysine ion pair in or near the second subsite has been proposed to enolize biotin prior to carboxylation by oxaloacetate (P. V. Attwood and W. W. Cleland, 1986, Biochemistry 25, 8197-8205). Enzyme modified with 2 equivalents of isoindole retained only 7% of the oxamate-induced, ADP/Pi-independent oxaloacetate decarboxylase activity, suggesting that there is at least one essential cysteine-lysine ion pair at or near the second subsite.

Carbamyl phosphate synthetase I (CPS I; EC6,3,4,16) is an autosomal recessive disorder characterized by hyperammonemia. We studied the molecular bases of CPS I deficiency in a newborn Japanese girl with consanguineous parents. Northern and Western blots revealed a marked decrease in CPS I mRNA and enzyme protein but with a size similar to that of the control, respectively. Sequencing of the patient's cDNA revealed a nine-nucleotide deletion at position 832-840. Sequencing analysis of the genomic DNA revealed a G to C transversion at position 840, the last nucleotide of an exon in the splice donor site. This substitution altered the consensus sequence of the splice donor site and the newly cryptical donor site in the exon caused the 9-bp in-frame deletion. This report seems to be the first complete definition of CPS I deficiency, at the molecular level.

In previous studies a Glu841-->Lys replacement in the carbamate phosphorylating domain located in the COOH half of the synthetase subunit of Escherichia coli carbamyl phosphate synthetase was shown to reduce overall synthesis of carbamyl phosphate by 4 orders of magnitude with either glutamine or NH3 as nitrogen donor (Guillou et al., 1992). In the present study, the mutant enzyme has been further analyzed for its glutamine hydrolytic activity. The glutaminase activity of the mutant enzyme has the following properties. (1) In the absence of other substrates the turnover number is only marginally different from that of the wild-type complex. (2) The Km for glutamine is 60 times higher than in wild-type complex and three times higher than in the separated glutaminase subunit. (3) In the present study wild-type carbamyl phosphate synthetase has been shown to catalyze glutamine hydrolysis by a mechanism involving an enzyme-bound acyl ester intermediate (gamma-glutamyl thioester). This intermediate is formed and is hydrolyzed with rates consistent with overall glutamine hydrolysis. At physiological concentrations of glutamine (1.2 mM), the steady-state concentration of gamma-glutamyl thioester is 0.3 mol/mol of wild-type enzyme. Under the same conditions, only 0.02 mol of thioester is measured in the mutant enzyme. Maximal accumulation of this covalent intermediate by the mutant enzyme required 10 times higher concentrations of free glutamine. (4) The rate of reaction with 2-amino-4-oxo-5-chloropentanoate, a glutamine analog known to specifically alkylate an active site cysteine residue, is 2 orders of magnitude slower in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

INTRODUCTION: N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. CASE REPORT: We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. CONCLUSION: NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

E. coli carbamyl phosphate synthetase binds 0.2-0.4 mol equivalents of glutamine in an acid resistant form. The bound material is quantitatively released as glutamate by weak base hydrolysis and as a mixture of 12% glutamate, 10% gamma-glutamylhydroxamate, and 70% pyrrollidonecarboxylic acid by hydrolysis with hydroxylamine. These results provide direct evidence for a gamma-glutamyl acyl ester on the enzyme. The absence of the acyl ester in a mutant carbamyl phosphate synthetase with a Cys269-->Ser substitution in the glutaminase subunit further suggests that the covalent intermediate is a thioester of Cys269. Under equilibrium conditions, the Cys269Ser mutant enzyme binds glutamine with a Kd of 7 +/- 1 microM, indicating that Cys269 is essential for acyl ester formation but not for binding of glutamine.

Summary AlgR is a transcriptional regulator of mucoidy in Pseudomonas aeruginosa, a critical virulence factor expressed in cystic fibrosis. AlgR belongs to the superfamily of bacterial signal transduction systems, and has been shown to bind to the algD promoter, a critical point in the regulation of mucoidy. This protein, like other typical response regulators, contains highly conserved residues known to be critical for the phosphorylation and signal transduction processes. However, a typical second component Interacting with AlgR has not been identified. Here we demonstrate that AlgR undergoes phosphorylation in vitro when interacting with the well-characterized histidine protein kinase CheA. These results Indicate that AlgR is capable of undergoing phosphorylation typical of other two-component signal transduction systems. Moreover, the phosphotransfer reaction between CheA and AlgR was found to be affected by the presence of carbamoyl phosphate, acetyl phosphate, and salts of phosphoramidic acid, recently shown to act as small-molecular-weight phospho-donors in the process of phosphorylation of several response regulators. These findings suggest that AlgR may react with intermediary metabolites such as carbamoyl phosphate and acetyl phosphate, and that these processes may play a role in the control of mucoidy in P. aeruginosa.

Valproic acid induced coma is presented in an adult patient without a history of metabolic disease. Liver biopsy revealed a reduction in activity of carbamyl phosphate synthetase-I, an enzyme obligated for transformation of ammonia to urea in the urea cycle. After recovery CT scan follow-up showed marked cerebral atrophy which did not exist prior to the state of coma. Risk factors are discussed.

13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase. For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect. This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate. The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers. These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity. Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random. With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate. The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.

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.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic isotope effects at the amide nitrogen of carbamyl phosphate were measured to determine whether catalysis by aspartate transcarbamylase proceeds through a cyanic acid or tetrabedral intermediate. Decomposition of the mono- and dianion of carbamyl phosphate was used as models for reaction of carbamyl phosphate proceeding through a tetrahedral adduct or cyanic acid intermediate, respectively. The 15 N kinetic isotope effects for decomposition of the mono- (1.0028) and dianion (1.0105) of carbamyl phosphate were sufficiently different to permit a distinction to be made between a cyanic acid intermediate or tetrahedral adduct

Aspartate-162 in the catalytic chain of aspartate transcarbamoylase is conserved in all of the sequences determined to date. The X-ray structure of the Escherichia coli enzyme indicates that this residue is located in a loop region (160's loop) that is near the interface between two catalytic trimers and is also close to the active site. In order to test whether this conserved residue is important for support of the internal architecture of the enzyme and/or involved in transmitting homotropic and heterotropic effects, the function of this residue was studied using a mutant version of the enzyme with an alanine at this position (Asp-162----Ala) created by site-specific mutagenesis. The Asp-162----Ala enzyme exhibits a 400-fold reduction in the maximal observed specific activity, approximately 2-fold and 10-fold decreases in the aspartate and carbamoyl phosphate concentrations at half the maximal observed specific activity respectively, a loss of homotropic cooperativity, and loss of response to the regulatory nucleotides ATP and CTP. Furthermore, equilibrium binding studies indicate that the affinity of the mutant enzyme for CTP is reduced more than 10-fold. The isolated catalytic subunit exhibits a 660-fold reduction in maximal observed specific activity compared to the wild-type catalytic subunit. The Km values for aspartate and carbamoyl phosphate for the Asp-162----Ala catalytic subunit were within 2-fold of the values observed for the wild-type catalytic subunit. Computer simulations of the energy-minimized mutant enzyme indicate that the space once occupied by the side chain of Asp-162 may be filled by other side chains, suggesting that Asp-162 is important for stabilizing the internal architecture of the wild-type enzyme.

Hyperammonemia, abnormalities in plasma amino acids and abnormalities of standard liver functions were corrected by orthotopic liver transplantation in a 14-day-old boy with carbamyl phosphate synthetase-I deficiency and in a 35-yr-old man with argininosuccinic acid synthetase deficiency. The first patient had high plasma glutamine levels and no measureable citrulline, whereas citrulline values were markedly increased in Patient 2. Enzyme analysis of the original livers showed undetectable activity of carbamyl phosphate synthetase-I in Patient 1 and arginosuccinic acid synthetase in Patient 2. Both patients were comatose before surgery. Intellectual recovery of patient 1 has been slightly retarded because of a brain abscess caused by Aspergillus infection after surgery. Both patients are well at 34 and 40 mo, respectively, after surgery. Our experience has shown that orthotopic liver transplantation corrects the life-threatening metabolic abnormalities caused by deficiencies in the urea cycle enzymes carbamyl phosphate synthetase-I and arginosuccinic acid synthetase. Seven other patients–six with ornithine transcarbamylase deficiency and another with carbamyl phosphate synthetase-I deficiency–are known to have been treated elsewhere with liver transplantation 1 1/2 yr or longer ago. Four of these seven recipients also are well, with follow-ups of 1 1/2 to 5 yr. Thus liver transplantation corrects the metabolic abnormalities of three of the six urea cycle enzyme deficiencies, and presumably would correct all. (Hepatology 1992;15:419–422).

Reye’s Syndrome (RS) was first documented by Douglas Reye and colleagues in 1963 as an “encephalopathy with fatty degeneration of the viscera,” a rare entity, but one that at that time was associated with an 80% mortality rate (Reye et al., 1963). Although the apparent incidence and outcome of the disease have improved, it continues to be one of the major causes of noninflammatory neurologic death after a viral illness in children (Sullivan-Boylai and Corey, 1981; Heubi et al., 1987).

Carbamyl-P:glucose and PPi:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] less than or equal to 0.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteretic behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 microM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P:glucose or PPi:glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentrations of glucose-6-P.

Two female siblings were treated for acute neonatal hyperammonaemia due to complete carbamyl phosphate synthetase I deficiency. The first child was detected clinically at 65 hours of age and therapy started at 79 hours. The second child was followed from birth and therapy started at 5 hours of age. The extrapolated rate of increase of blood ammonia, in the first hours of life before therapy started, was 19 mumol L-1 h-1 in both babies. Peak blood ammonia level was 2235 mumol/L in the first (clinically detected) child and 271 mumol/L in the second (prospectively followed) child. The second child became symptomatic at 3 hours of age when blood ammonia level was as low as 90 mumol/L, whereas blood ammonia levels above 100 mumol/L caused no symptoms during recovery. The child detected clinically required haemodialysis and peritoneal dialysis to treat the hyperammonaemia. In the prospectively treated child, early therapy with intravenous sodium benzoate and sodium phenylacetate slowed the rate of increase in blood ammonia level, but this therapy did not prevent the need for peritoneal dialysis.

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)

In a reaction that is analogous to the phosphorylation of ADP from carboxyphosphate, pyruvate carboxylase catalyses the formation of ATP from carbamoyl phosphate and ADP at a rate that is about 0.3% of the pyruvate-carboxylation reaction and about 3% of the full reverse reaction. Acetyl-CoA stimulates the phosphorylation of ADP from carbamoyl phosphate but is not an essential requirement of the reaction. Mg2+ also stimulates the reaction, and in the range of Mg2+ concentrations considered the effect of V is much larger in the absence of acetyl-CoA than in its presence. Acetyl-CoA and Mg2+ may be acting in a co-operative way to stimulate the phosphorylation of ADP in a similar way to their effects on the pyruvate-carboxylation reaction. The phosphorylation of ADP by carbamoyl phosphate is also stimulated by the presence of biotin in the part of the active site where this reaction occurs, but again it is not absolutely required for the reaction to proceed. The pH profiles of the phosphorylation of ADP by carbamoyl phosphate indicate that there are at least two ionizable residues involved in the reaction, one of which probably has a role in the release of carbamate from the active site.

Lactobacillus plantarum is often found in nutrient-rich habitats with elevated levels of inorganic carbon (IC), and IC-dependent growth is commonly encountered in natural isolates of this species. High CO(2)-requiring (HCR) prototrophs are unable to grow under conditions of low IC unless arginine and pyrimidines are provided. Prototrophy is restored under high IC conditions, that is in 4% CO(2)-enriched air or bicarbonate-supplemented medium. Bicarbonate is required for the synthesis of carbamoyl phosphate (CP), a precursor of both arginine and pyrimidine biosynthesis. We hypothesize that at low IC levels, intracellular CP pools limit growth through the limitation of arginine and nucleotide supplies. HCR mutants obtained in the laboratory can be classified into 3 functional groups: mutants with impaired CP synthesis, increased CP consumption or increased CP requirements relative to wild type. This classification provides a framework for investigating the origin of the HCR phenotype in natural environmental isolates of Lactobacillus species, and to investigate the hypothesis that a low level of carbamoyl phosphate is a major determinant of the CO(2)-dependent growth phenotype often observed in L. plantarum isolates. Copyright (c) 2008 S. Karger AG, Basel.

Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme. In the present work, this residue was replaced by an asparagine through site-directed mutagenesis. The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate. In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group. In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3). In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type.

Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM) for the pathogenic amastigote form in in vitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials.

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.

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.

In this paper it is shown that the adsorption of 5'-adenosine monophosphate (5'-AMP) onto precipitated calcium phosphate exhibits a sigmoidal profile as revealed by isotherms at 45 degrees C. This result indicates a cooperative behavior in the adsorption of 5'-AMP. The relationship between adsorption capacity and surface area of the sedimented matrix may be interpreted as an indication that there is a monolayer of the absorbed nucleotide on the solid surface. The pH dependence of adsorption suggests that the negatively charged phosphoryl group of 5'-AMP interacts with a positively charged site (possibly Ca2+) on the matrix surface. The adsorption of the nucleotide is markedly decreased at pH values above 8.0. The Dixon-like plot of the effect of pH suggests an inhibitory role of hydroxyl ions in the adsorption of 5'-AMP. At pH 7.5, other anions such as pyrophosphate, tripolyphosphate and carbamyl phosphate also inhibit the adsorption of the nucleotide, probably by interacting with its adsorption site. We suggest that these phosphorylated molecules could have played a role in chemical evolution by modulating the amount of nucleotides adsorbed onto mineral surfaces. The significance of these phenomena in chemical evolution is discussed.

We have investigated the putative carbamylphosphate- and ornithine-binding domains in ornithine transcarbamylase from rat liver using site-directed mutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-X and therefore implicated in the binding of the phosphate moiety of carbamylphosphate has been replaced with a leucine. This results in a dramatic reduction of catalytic activity, although the enzyme is synthesized in cells stably transfected with the mutant clone and imported, correctly processed and assembled into a homotrimer in mitochondria. The sole cysteine residue (Cys271) has been implicated in ornithine binding by the chemical modification studies of Marshall and Cohen in 1972 and 1980 (J. Biol. Chem., 247, 1654-1668, 1669-1682; 255, 7291-7295, 7296-7300). Replacement of this residue with serine did not eliminate enzyme activity but affected the Michaelis constant for ornithine (Kb), increasing it 5-fold from 0.71 to 3.7 mM and reduced the kcat at pH 8.5 by 20-fold. These changes represent a loss in apparent binding energy for the enzyme--ornithine complex of 2.9 kcal/mol, suggesting that Cys271 is normally involved in hydrogen bonding to the substrate, ornithine. The cysteine to serine substitution also caused the dissociation constant (Kii) for the competitive inhibitor, L-norvaline to be increased 10-fold, from 12 to 120 microM. The small loss in binding energy and relatively high residual catalytic activity of the mutant strongly suggests that a number of other residues are involved in the binding of ornithine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphonacetyl-L-aspartate (PALA) is a rationally-synthesized analog of the transition-state intermediate in the formation of carbamyl aspartate from carbamyl phosphate and aspartic acid by aspartate carbamyl transferase (ACTase). PALA is thus a potent inhibitor of the enzyme (Ki about 10(-8) M for ACTases of various origins), which in whole cells blocks the de novo synthesis of pyrimidines. In vivo, low doses of PALA inhibit whole body pyrimidine synthesis. While this action is cytotoxic in vitro, extensive human testing demonstrates that PALA alone is devoid of selective antitumor activity. Recent interest in the therapeutic action of PALA derives from the demonstration that its action potentiates the cytotoxicity of several cytotoxic drugs, notably 5-fluorouracil (5-FU). Results from clinical trials of PALA and 5-FU in combination in colorectal cancer suggest that biochemical modulation with regimens which follow the principles determined in preclinical studies may enhance the efficacy of current therapy.

Background Hyperammonemia in patients with methylmalonic aciduria (MMA) and propionic aciduria (PA) is caused by accumulation of propionyl-CoA which decreases the synthesis of N-acetyl-glutamate, the natural activator of carbamyl phosphate synthetase 1. A treatment approach with carglumic acid, the structural analogue of N-acetyl-glutamate, has been proposed to decrease high ammonia levels encountered in MMA and PA crises. Case presentation We described two patients (one with MMA and one with PA) with hyperammonemia at diagnosis. Carglumic acid, when associated with standard treatment of organic acidurias, may be helpful in normalizing the ammonia level. Conclusion Even though the usual treatment which decreases toxic metabolites remains the standard, carglumic acid could be helpful in lowering plasma ammonia levels over 400 micromol/L more rapidly.

Ammonia is produced by amino acid and protein catabolism and is toxic to brain and muscles. It is converted to urea in the liver by urea-cycle enzymes, which is then excreted through the kidneys. Hyperammonaemia may result from a genetic defect or deficiency of the urea-cycle enzymes, or acquired conditions such as Reye's syndrome, liver failure, high-dose chemotherapy and severe infection [1]. In the urea cycle, which takes place exclusively in periportal hepatocytes, six enzymes are involved in the conversion of ammonia to urea. These areN-acetylglutamate synthetase, carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase. A defect or deficiency of any of these enzymeswill result in hyperammonaemia [2]. Hyperammonaemia is most severe when the enzyme defect is in the early steps of the urea cycle such as carbamyl phosphate synthetase deficiency and ornithine transcarbamylase. Infection, trauma or ingestion of large amounts of proteins is usually the precipitating factor in causing hyperammonaemia. The clinical features of hyperammonaemia are usually nonspecific in adults and include vomiting, lethargy, sleep and behavioural disturbances, hallucinations, delusions and psychosis. Electroencephalogram may show an encephalopathic slow wave pattern [3]. Renal tubular acidosis (RTA) are a group of disorders of renal tubular function that lead to hyperchloremic metabolic acidosis in the presence of well-preserved glomerular function.

Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N-acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N-acetylornithine. Recently, a novel N-succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified. Structural comparisons of AOTCase from X. campestris and SOTCase from B. fragilis revealed that residue Glu92 (X. campestris numbering) plays a critical role in distinguishing AOTCase from SOTCase. Enzymatic assays of E92P, E92S, E92V, and E92A mutants of AOTCase demonstrate that each of these mutations converts the AOTCase to an SOTCase. Similarly, the P90E mutation in B. fragilis SOTCase (equivalent to E92 in X. campestris AOTCase) converts the SOTCase to AOTCase. Hence, a single amino acid substitution is sufficient to swap the substrate specificities of AOTCase and SOTCase. X-ray crystal structures of these mutants in complexes with CP and N-acetyl-L-norvaline (an analog of N-acetyl-L-ornithine) or N-succinyl-L-norvaline (an analog of N-succinyl-L-ornithine) substantiate this conversion. In addition to Glu92 (X. campestris numbering), other residues such as Asn185 and Lys30 in AOTCase, which are involved in binding substrates through bridging water molecules, help to define the substrate specificity of AOTCase. These results provide the correct annotation (AOTCase or SOTCase) for a set of the transcarbamylase-like proteins that have been erroneously annotated as ornithine transcarbamylase (OTCase, EC 2.1.3.3).

We assessed the possible upregulation of glutamine synthetase (GS) and typical ‘fish type’ carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish ( Clarias batrachus ) during exposure to 25 mM NH 4 Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine–urea cycle possibly through the involvement of cortisol.

Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature, the transition between T- and R-states involves significant changes in the quaternary structure, which were detected by analytical ultracentrifugation. The enzyme is allosterically regulated by ATP (activator) and by CTP and UTP (inhibitors). Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas ATP and UTP act at millimolar concentrations, CTP inhibits at micromolar concentrations, suggesting that in P. abyssi, inhibition by CTP is the major control of enzyme activity. While V(max) increased with temperature, cooperative and allosteric effects were little or not affected, showing that molecular adaptation to high temperature allows the flexibility required to form the appropriate networks of interactions. In contrast to the same enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the carbamyl phosphate analogue phosphonacetate; this difference supports the idea that in native cells ATCase interacts with carbamyl phosphate synthetase to channel the highly thermolabile carbamyl phosphate.

Carbamyl phosphate synthase deficiency (CPS) is a rare urea cycle defect. We present a case of a 41-year-old woman diagnosed with CPS deficiency during pregnancy. She is the oldest CPS-deficient patient, at diagnosis, reported to date and the first to be diagnosed during pregnancy. This case highlights the need for consideration of inborn errors of metabolism in adults presenting with unusual neurological and psychiatric conditions.

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 °C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of l-histidine to l-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 °C was compared to the same strain grown at 37 °C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 °C and reduced at 4 and 37 °C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC.

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.

The structure of aspartate transcarbamylase of Escherichia coli ligated to products (phosphate and N-carbamyl-l-aspartate) has been determined at 2.37 A resolution (R-factor = 0.23, R(free) = 0.27). Results might indicate a product release mode, rather than close analogues to the transition state like those found in our earlier studies of other ligands (N-phosphonacetyl-L-aspartate, carbamyl phosphate plus malonate, phosphonoacetamide plus malonate, or citrate plus phosphate). Ordered product release, first carbamylaspartate (CLA) and then phosphate, might be facilitated by a 4 A movement of phosphate from the substrate-analogue position to the product (phosphate) binding position, and by a somewhat similar release movement of the other product (CLA) relative to its analogue (citrate). This movement is consistent with earlier studies of binding of either pyrophosphate or phosphate alone [Honzatko, R. B., and Lipscomb, W. N. (1982) J. Mol. Biol. 160, 265-286].

Structures of the R-state of Escherichia coli ATCase maintained with carbamyl phosphate and succinate, phosphonoacetamide and malonate, or N-phosphonacetyl-L-aspartate (PALA) have previously been made in the space group P321, in which the two independent r (regulatory) and two independent c (catalytic) chains are repeated by crystallographic symmetry to yield the holoenzyme c 6 r 6 , ((c 3 ) 2 (r 2 ) 3 ). The exploration of a new crystalline R-state P2 1 2 1 2 1 was undertaken to examine the c 3 ...c 3 expansion of 11 A in the T-to-R transition, and to further test whether intermolecular contacts influence the binding of PALA. The results show that the expansion along the 3-fold axis is 10 A, and that the binding modes of the six crystallographic independent PALA molecules are virtually identical to one another, and to modes described previously. As further test, the PALA, a bisubstrate analogue, was displaced by citrate and phosphate, where citrate is an analogue of product carbamylaspartate. The results support the conclusions about the binding of the three previously studied analogues, and further support, within about 0.5 A, the structure proposed for the transition state [Gouaux, J. E., Krause, K. L., and Lipscomb, W. N. (1987) Biochem. Biophys. Res. Commun. 142, 893-897; Jin, L., Stec, B., Lipscomb, W. N., and Kantrowitz, E. R. (1999) Proteins: Struct., Funct., Genet. 37, 729-742].

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

The synthesis of PRPP from ATP and ribose-5-phosphate is catalyzed by phosphoribosylpyrophosphate synthetase as follows