Amino acid sequence of rat argininosuccinate lyase deduced from cDNA

Argininosuccinate lyase [EC 4.3.2.1] is an enzyme of the urea cycle in the liver of ureotelic animals. The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner. The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M. (1986) Biochem. Int. 13, 433-438), was determined. The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp. The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme. The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus. However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes. Therefore, the human sequence should be re-examined. Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.     

Molecular cloning of cDNA for argininosuccinate lyase of rat liver

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.     

Evolutionary aspects of urea cycle enzyme genes

The functions and expression pattern of urea cycle enzymes have undergone considerable changes during the course of evolution. Sequence analyses shows that urea cycle enzymes from mammals are homologous to microbial enzymes of the arginine-metabolic pathway. Recently, an unexpected relationship was found between argininosuccinate lyase (EC 4.3.2.1), the fourth enzyme of the cycle, and delta-crystallin, a lens structural protein of birds and reptiles.     

Inhibition of urea cycle enzymes by lysine and saccharopine

Crude and purified preparations of argininosuccinate synthetase, argininosuccinate lyase and arginase were subjected to inhibition studies with L-lysine and saccharopine. Saccharopine proved to be the more potent inhibitor of argininosuccinate synthetase and lyase, whereas lysine had more effect on arginase. Similar results were found with pure enzyme and crude preparations. Computer analysis of the results suggested that inhibition of urea cycle enzymes by saccharopine and lysine might have contributed to the high levels of citrulline found in a human patient with saccharopinuria, a defect of saccharopine metabolism, but that this was unlikely to be the sole explanation.     

Modulation of intestinal urea cycle by dietary spermine in suckling rat

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure. Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity.

delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.     

Aspartase/fumarase superfamily: a common catalytic strategy involving general base-catalyzed formation of a highly stabilized aci-carboxylate intermediate

Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.     

The structure of adenylosuccinate lyase, an enzyme with dual activity in the de novo purine biosynthetic pathway

Background: Adenylosuccinate lyase is an enzyme that plays a critical role in both cellular replication and metabolism via its action in the de novo purine biosynthetic pathway. Adenylosuccinate lyase is the only enzyme in this pathway to catalyze two separate reactions, enabling it to participate in the addition of a nitrogen at two different positions in adenosine monophosphate. Both reactions catalyzed by adenylosuccinate lyase involve the beta-elimination of fumarate. Enzymes that catalyze this type of reaction belong to a superfamily, the members of which are homotetramers. Because adenylosuccinate lyase plays an integral part in maintaining proper cellular metabolism, mutations in the human enzyme can have severe clinical consequences, including mental retardation with autistic features. Results: The 1.8 A crystal structure of adenylosuccinate lyase from Thermotoga maritima has been determined by multiwavelength anomalous dispersion using the selenomethionine-substituted enzyme. The fold of the monomer is reminiscent of other members of the beta-elimination superfamily. However, its active tetrameric form exhibits striking differences in active-site architecture and cleft size. Conclusions: This first structure of an adenylosuccinate lyase reveals that, along with the catalytic base (His141) and the catalytic acid (His68), Gln212 and Asn270 might play a vital role in catalysis by properly orienting the succinyl moiety of the substrates. We propose a model for the dual activity of adenylosuccinate lyase: a single 180 degrees bond rotation must occur in the substrate between the first and second enzymatic reactions. Modeling of the pathogenic human S413P mutation indicates that the mutation destabilizes the enzyme by disrupting the C-terminal extension.     

Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase

The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.     

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, alpha-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2-14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of alpha-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.     

Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase.

Adenylosuccinate synthetase (AS) catalyzes the first committed step in the conversion of IMP to AMP. A cDNA was isolated from a human liver library which encodes a protein of 455 amino acids (M(r) of 49,925). Alignments of human, mouse, Dictyostelium discoideum and E. coli AS sequences identify a number of invariant residues which are likely to be important for structure and/or catalysis. The human AS sequence was also 19% identical to the human urea cycle enzyme, argininosuccinate synthetase (ASS), which catalyzes a chemically similar reaction. Both human liver and HeLa AS mRNA showed signals of 2.3 and 2.8 kb. An unmodified N-terminus is required for function of the human AS enzyme in E. coli mutants lacking the bacterial enzyme. The human cDNA provides a means to assess the possible role of AS abnormalities in unclassified, idiopathic cases of gout.     

Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: An immunocytochemical study

Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.     

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts

There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of L-citrulline from L-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 microM), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.     

Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin

Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.     

Relative argininosuccinate synthetase mRNA levels and gene copy number in canavanine-resistant lymphoblasts

Mutants resistant to the arginine analogue, canavanine, have been isolated from two normal lymphoblast lines, MGL8B2 and MGL33. These mutants constitutively express up to 200-fold higher amounts of structurally normal argininosuccinate synthetase, the urea cycle enzyme that converts citrulline to argininosuccinate. Relative levels of argininosuccinate synthetase mRNA were compared among normal and canavanine-resistant lines using in vitro translation of poly(adenylic acid) RNA and blot hybridization of total cytoplasmic RNA to an argininosuccinate synthetase cDNA. Both of these approaches indicated that the canavanine-resistant lines contain increased steady-state levels of synthetase-specifc mRNA relative to their sensitive parents and that these were roughly correlated with levels of enzyme activity. Blot hybridization of Eco RI-digested genomic DNA preparations revealed no detectable differences in argininosuccinate synthetase structural gene copy number between normal and canavanine-resistant lymphoblasts, demonstrating that the canavanine-resistant phenotype is not caused by gene amplification.     

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.

The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.