Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.

Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation.

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.

Inhibition of carbamyl phosphate synthetase-I and glutamine synthetase by hepatotoxic doses of acetaminophen in mice.

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS-PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.

Photoaffinity labeling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor.

UMP is a highly specific reagent for photoaffinity labeling of the allosteric inhibitor site of carbamyl phosphate synthetase (CPS) from Escherichia coli and has been found to be photoincorporated in the COOH-terminal domain of the large subunit [Rubio et al. (1991) Biochemistry 30, 1068-1075]. In the present work we identify lysine 992 as the residue that is covalently attached to UMP. This identification is based on two lines of evidence. First, [14C]UMP is found to be incorporated between residues 939 and 1006, as shown by peptide mapping and by mass estimates of [14C]UMP-peptides generated by chemical and enzymatic cleavage of CPS. Secondly, we have purified two radioactive peptides derived exclusively from those enzyme molecules (approximately 5% of the total enzyme) that had incorporated [14C]-UMP. Edman analyses show the sequences of the labeled peptides (989)LVNXVHEGRPHIQD and (989)LVNXVHE to be overlapping. Since neither a phenylthiohydantoin (Pth) derivative (in cycle 4) nor any radioactivity is released from the membrane during sequencing, we can conclude that Lys992 and [14C]-UMP form a covalent adduct that remains bound to the membrane. Formation of this adduct agrees with all of the evidence and with the finding that UMP labeling prevents trypsin cleavage at Lys992. Lysine 992 is invariant in those CPSs that are inhibited by UMP, and is located 30 residues upstream of the site whose phosphorylation in hamster CAD reduces inhibition of CAD by UTP. Multiple sequence alignment of the residues surrounding Lys992 of the E. coli enzyme and the corresponding residues of the yeast and animal enzymes supports the existence of a uridine nucleotide binding fold in this region of the protein. We conclude that sequence changes in the binding fold provide a structural basis for the different regulatory properties found among CPSs I, II, and III.

Purification and characterization of carbamoyl-phosphate synthetase from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi.

Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the carbamoyl-phosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor, whose duplication gave the genes coding for carbamoyl-phosphate synthetases and carbamate kinases.

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl-phosphate synthetase I. Studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine.

The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region.

The interaction of cardiolipin with rat liver carbamoyl phosphate synthetase I.

A selective interaction of rat liver carbamoyl phosphate synthetase I with cardiolipin, and other anionic phospholipids, has been demonstrated. The enzymatic activity of the synthetase is inhibited by cardiolipin and, to a lesser extent, by phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. This group of anionic phospholipids also induced a conformational change in the synthetase, yielding a species with increased exposure of the linkages between independently folded domains of the enzyme, as determined by limited proteolysis under nondenaturing conditions. The interaction of cardiolipin with carbamoyl phosphate synthetase I was a fairly slow process, with complex kinetics, and was apparently irreversible. The inclusion of Mg2+ or of MgATP in the incubation mixture prevented the cardiolipin effects. The zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine had negligible effects on the structure and activity of the synthetase. This interaction between cardiolipin and carbamoyl phosphate synthetase I potentially constitutes one of the mechanisms by which the synthetase forms its loose association with the inner mitochondrial membrane. Multiple mechanisms, including synthetase conformational changes, cardiolipin phase changes, and ATP/ADP binding site involvement, are possibly involved in the phospholipid/synthetase interaction and the resulting potential regulatory mechanism(s) for urea cycle activity.

Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.

Inhibition of carbamoyl phosphate synthetase-I by dietary dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), administered per os, serves to prevent or retard the development of a variety of genetic and induced disorders in mice and rats. This treatment also results in the development of hepatomegaly, a change of liver color from pink to mahogany, peroxisome proliferation in hepatocytes and alterations in hepatocyte mitochondria morphology and respiration. We used one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to identify changes in the relative levels of liver proteins produced by DHEA treatment of rodents. In mouse liver, there were apparent increases in the levels of 26 proteins and decreases in the levels of 7 proteins. Of the induced proteins the most prominent had Mr approximately 72 K; this protein was identified in a previous study as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Another protein of Mr approximately 28 K, of unknown nature, also was induced markedly by DHEA treatment of mice and rats. A protein of Mr approximately 160 K, which was identified as carbamoyl phosphate synthetase-I (CPS-I), was decreased markedly by DHEA action. This enzyme, which comprises approx. 15-20% of mitochondrial matrix protein, is involved in the entry and rate-limiting step of the urea cycle. The specific activity of CPS-I also was significantly decreased by DHEA, but serum urea levels were normal. To determine whether steroids other than DHEA also induced similar changes, mice were treated with various steroids for 14 days and, thereafter, liver proteins were evaluated by SDS-PAGE: estradiol-17 beta and isoandrosterone induced both the approximately 72 and approximately 28 kDa proteins, testosterone and androsterone induced the 28 kDa protein only, but etiocholanolone, pregnenolone and progesterone were without effect. The findings of this study serve to demonstrate that: (i) hepatic protein levels are affected by DHEA treatment of mice and rats; (ii) liver CPS-I activity is decreased significantly by DHEA treatment, but serum urea levels remain within the normal range; and (iii) sex steroids and some of their precursors, when administered per os, also alter liver protein levels.

A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I.

The requirements for binding at the N-acetyl-L-glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the alpha-carbon, enhances binding 5000-fold. The subsite for the delta-carboxyl is mobile with respect to that for the alpha-carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, indicating a strong 'chelate' effect. Substituents revealed the existence of steric constraints around the delta-carboxyl, the alpha and gamma-carbons, and the whole of the acetamido group. However, phenyl substituents at the beta-carbon did not hamper binding, indicating that substituents at the beta-carbon face the solution. This is consistent with binding of acetylglutamate as the minimum-energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low-affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the inhibitors but it is crucial for the binding of the activators, and the high- and low-affinity conformations of the site differ markedly in structural selectivity.

Hepatic ATP content and hyperammonemia induced by CCl4 in rats.

An investigation of the mechanism of development of hyperammonemia observed in CCl4-induced hepatic encephalopathy was performed in rats. CCl4 (1.0 ml/kg 3 times per week for over 10 weeks) caused a severe hyperammonemia and depletion of hepatic ATP contents in only those rats with hepatic encephalopathy. However, CCl4 (1.0 ml/kg 3 times per week for 7 weeks) did not cause hepatic encephalopathy and did not change in blood ammonia levels. Administration of 2,4-dinitrophenol (2,4-DNP) in these CCl4-treated rats caused hepatic encephalopathy within 30 min after injection and then the increase of 140 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. However, the administration of 2,4-DNP in CCl4-untreated rats did not cause hepatic encephalopathy within 30 min after injection although the increase of 70 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by 85% and 60% respectively, in rats treated with CCl4 plus 2,4-DNP. However, in rats treated with 2,4-DNP and without CCl4, the hepatic activities of CPS and ASS were inhibited only 25% and 0%, respectively. These findings suggest that the severe hyperammonemia, which may be produced by the decrease of hepatic ATP content and the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation.

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.

Partial characterization of intracellular protease activity which participates in the inactivation of carbamyl phosphate synthase in Phycomyces blakesleeanus.

We have partially characterized an intracellular fraction from Phycomyces blakesleeanus which shows proteolytic activity. The apparent thermal inactivation constant (Kd) was 0.12 min-1 at 50 degrees C. This proteolytic fraction was split into two active fractions by ultrafiltration using a membrane with an exclusion size of 30,000. Both fractions were inhibited by phenyl methyl sulphonyl fluoride. The Ki value for the fraction with molecular weight greater than 30,000 was 0.075 mM. The fraction with molecular weight less than 30,000 inactivated the Phycomyces CPS.

Blood ammonia levels and hepatic encephalopathy induced by CCl4 in rats.

An investigation of the mechanism of development of hepatic encephalopathy induced by CCl4 was performed in rats. CCl4 (1.0 ml/kg three times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. The blood ammonia levels were tremendously increased in only those rats with hepatic encephalopathy. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by CCl4. However, the causality between the inhibition of CPS or ASS activity and the increase in blood ammonia levels was not observed. On the other hand, the content of ATP, which is a substrate of CPS and ASS, was decreased by 60% in liver of rats with hepatic encephalopathy. The activity of Mg2+-ATPase which can decompose hepatic ATP was increased by 60 and 300% in mitochondria and microsomes, respectively, of livers of rats with CCl4-induced encephalopathy. There was a good correlation between the decreased hepatic ATP content and the increased mitochondrial Mg2+-ATPase activity. Furthermore, there was also a good correlation between the increase in blood ammonia levels and the increase in Mg2+-ATPase activity in microsomes. These findings suggest that hyperammonemia, which was produced by the decrease in hepatic content and by the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.

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.

Inhibition of carbamoyl-phosphate synthase (ammonia) by Tris and Hepes. Effect on Ka for N-acetylglutamate.

The apparent Ka for N-acetylglutamate of rat liver carbamoyl-phosphate synthase is 11 microM in phosphate buffer, a value 10-fold lower than reported in other buffer systems. Tris and Hepes inhibit competitively with N-acetylglutamate. The proportion of carbamoyl-phosphate synthase in the active enzyme-acetylglutamate complex in vivo may be higher than previous calculations suggest, which re-opens the question of the involvement of N-acetylglutamate in the regulation of urea synthesis.

Inhibition of ureogenesis in isolated rat liver cells by a non-steroidal anti-inflammatory drug (butibufen).

The effect of a non-steroidal anti-inflammatory drug (butibufen) on ureogenesis in isolated rat hepatocytes has been studied. Butibufen at 0.4 mM, and particularly at 2 mM, strongly inhibited urea synthesis. The drug at these concentrations also inhibited markedly carbamoylphosphate synthetase activity. In addition, 2 mM butibufen lowered ATP concentrations of the cells and enhanced oxygen consumption in isolated liver mitochondria. The results suggest that the inhibition by 0.4 mM butibufen on carbamoylphosphate synthetase activity can account for the entire inhibition of ureogenesis, whereas the decreased cellular ATP concentration at 2 mM butibufen might be at least partly responsible for low carbamoylphosphate synthesis and thus, for reduced urea production. The decrease in ATP levels probably results from uncoupling effects of butibufen on oxidative phosphorylation.