Nitro analogs of substrates for argininosuccinate synthetase and argininosuccinate lyase

The nitro analogs of aspartate and argininosuccinate were synthesized and tested as substrates and inhibitors of argininosuccinate synthetase and argininosuccinate lyase, respectively. The Vmax for 3-nitro-2-aminopropionic acid was found to be 60% of the maximal rate of aspartate utilization in the reaction catalyzed by argininosuccinate synthetase. Only the nitronate form of this substrate, in which the C-3 hydrogen is ionized, was substrate active, indicating a requirement for a negatively charged group at the beta carbon. The V/K of the nitro analog of aspartate was 85% of the value of aspartate after correcting for the percentage of the active nitronate species. The nitro analog of argininosuccinate, N3-(L-1-carboxy-2-nitroethyl)-L-arginine, was a strong competitive inhibitor of argininosuccinate lyase but was not a substrate. The pH dependence of the observed pKi was consistent with the ionized carbon acid (pK = 8.2) in the nitronate configuration as the inhibitory material. The pH-independent pKi of 2.7 microM is 20 times smaller than the Km of argininosuccinate at pH 7.5. These results suggest that the tighter binding of the nitro analog relative to the substrate is due to the similarity in structure to a carbanionic intermediate in the reaction pathway.     

Induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic AMP and dexamethasone

Arginine is an intermediate in the elimination of excess nitrogen and is the substrate for nitric oxide synthesis. Arginine synthesis has been reported in brain tissue. We have studied the activity of the arginine biosynthetic enzymes argininosuccinate synthetase and argininosuccinate lyase in dexamethasone and/or dibutyryl cyclic AMP treated rat astrocyte cultures. Argininosuccinate lyase activity was stimulated by treatment with either effector and an additive effect was obtained when both agents were added simultaneously. Argininosuccinate synthetase was also increased in dexamethasone treated astrocytes. The effect of dibutyryl cyclic AMP on argininosuccinate synthetase was variable, suggesting a role for additional factors in its regulation as compared to argininosuccinate lyase. Regulation of arginine synthesis in astrocytes may be important to insure that arginine is not limiting for nitric oxide synthesis in neural tissue.     

Kinetic mechanism of argininosuccinate synthetase

The kinetic mechanism of bovine liver argininosuccinate synthetase has been determined at pH 7.5. The initial velocity and product and dead-end inhibition patterns are consistent with the ordered addition of MgATP, citrulline, and aspartate, followed by the ordered release of argininosuccinate, MgPPi, and AMP. The mechanism is also in accord with the formation of citrulline-adenylate as a reactive intermediate [O. Rochovansky, and S. Ratner, (1967) J. Biol. Chem. 242, 3839-3849]. No evidence was obtained for nonlinear double-reciprocal plots with any of the three substrates.     

Homology of delta crystallin and argininosuccinate lyase

1. Delta crystallin, a major lens protein characteristic of birds and reptiles, is homologous to argininosuccinate lyase; 57% of the residues in chicken delta crystallin and human lyase are identical. 2. Even more similar (62% identical residues) to the human lyase is the sequence translated from the presumably inactive delta-2 gene of the delta crystallin locus. 3. As both delta crystallin and lyase are synthesized in birds only during the embryonic and juvenile stages, the persistence of delta crystallin in the adult lens appears to be paedomorphic. 4. Possible correlations of the origins of delta crystallin with other events in sauropsid evolution are proposed.     

Sequence for human argininosuccinate synthetase cDNA.

The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression.     

Stereochemical probes of the argininosuccinate synthetase reaction

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Acid-base catalysis in the argininosuccinate lyase reaction

The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.     

Substrate-induced inactivation of argininosuccinate lyase by monofluorofumarate and difluorofumarate

Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 degrees C, and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 microM and 0.5%. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min-1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.     

New radioisotopic assays of argininosuccinate synthetase and argininosuccinase.

Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.     

Determination of argininosuccinate in normal blood serum and liver

Argininosuccinate has been determined in normal serum and liver extract of rats by chromatographic analysis on Dowex-1-acetate after a brief period of in vivo labeling with l-[U-¹⁴C] citrulline. The amounts found were within the range of other amino acid intermediates of the ornithine cycle, determined in the same samples. Both ¹⁴C and ninhydrin color were measured. Chromatography on Amberlite columns, subsequent to fractionation on Dowex, allowed determination of the other amino acids. Fractionation on Dowex-1-acetate provides a reliable and highly selective method for the analysis of minute amounts of argininosuccinate in biological samples containing other amino acids.     

Isotopic probes of the argininosuccinate lyase reaction

The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotopic substitution at the reaction centers. A primary deuterium isotope effect of 1.0 on both V and V/K is obtained with (2S,3R)-argininosuccinate-3-d, while a primary 15N isotope effect on V/K of 0.9964 +/- 0.0003 is observed. The 15N isotope effect on the equilibrium constant is 1.018 +/- 0.001. The proton that is abstracted from C-3 of argininosuccinate is unable to exchange with the solvent from the enzyme-intermediate complex but is rapidly exchanged with solvent from the enzyme-fumarate-arginine complex. A deuterium solvent isotope effect of 2.0 is observed on the Vmax of the forward reaction. These and other data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the inverse 15N primary isotope effect and the solvent deuterium isotope effect suggest that protonation of the guanidino group and carbon-nitrogen bond cleavage of argininosuccinate are kinetically significant.     

Messenger RNA coding for argininosuccinate synthetase in citrullinemia

Messenger RNA coding for argininosuccinate synthetase (ASS), extracted from the livers of some patients with citrullinemia, was analyzed using a cell-free translation system and dot and Northern blot hybridization with cDNA probe for ASS. In patients with quantitative-type citrullinemia, called type II here, previous studies have demonstrated that the hepatic content of the enzyme was about 10% of the control value, whereas the translatable mRNA level for the enzyme was similar to that of control livers. Here, we confirmed that the type II liver contained an almost normal amount of mRNA coding for ASS, judged by the dot-blot hybridization technique with cDNA. Northern blot hybridization of RNA indicated that there was hybridizable mRNA of approximately normal size (about 1.7 kilobase [kb]) in each, suggesting that large structural gene deletions had not occurred. These results indicate that in type II citrullinemia, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased or inhibited translation in the liver. Another type of citrullinemia was found and classified as type III. It is characterized by no detectable enzyme activity for ASS or translation activity for ASS mRNA. However, a smaller amount of RNA molecule hybridized for ASS cDNA was detected.     

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.Subject terms: Cancer metabolism, Regeneration, Cancer metabolism, Experimental models of disease, Oncogenesis