Determination of the mechanism of the argininosuccinate synthetase reaction by static and dynamic quench experiments

The reactions catalyzed by argininosuccinate synthetase have been examined by the use of static and dynamic quench techniques. The time course of the forward reaction (22 degrees C) at pH 8.0 is characterized by a "burst" of AMP formation upon quenching with acid that is equivalent to 0.59 mol of enzyme. The pre-steady-state rate is followed by a slower steady-state rate of 0.60 s-1. The rate constant for the transient phase is 9.7 s-1. The time course for the formation of argininosuccinate is linear and shows neither a "lag" nor a burst phase. These results have been interpreted to mean that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. In the presence of aspartate the rate constant for the formation of citrulline adenylate (6.2 s-1) from ATP and citrulline is 7 times faster than the rate of formation of argininosuccinate from aspartate and citrulline adenylate (0.9 s-1). This suggests that the second step is predominantly rate limiting. The rate constant for the formation of citrulline adenylate in the absence of enzyme-bound aspartate (0.01 s-1) is 600 times slower than when aspartate is present. This indicates that the binding of aspartate to the enzyme regulates the formation of the intermediate. These results are in complete accord with our previously published steady-state kinetic scheme showing sequential addition of substrates.     

Control of argininosuccinate synthetase by arginine in human lymphoblasts

Human lymphoblasts in long-term culture have the enzyme activities necessary to convert citrulline to arginine: argininosuccinate synthetase and argininosuccinate lyase. Upon transfer from arginine-supplemented to citrulline-supplemented medium, lymphoblasts exhibit a lag period before resuming exponential growth. During this lag the specific activity of argininosuccinate synthetase increases an average of 60-fold. Argininosuccinate lyase activity remains unchanged. If normal lymphoblasts are starved in arginine-deficient medium without citrulline or if argininosuccinate lyase--deficient lymphoblasts are transferred to citrulline-containing medium, argininosuccinate synthetase activity increases linearly for several days and reaches even higher levels. Cycloheximide blocks the increase in enzyme activity. Cells grown in citrulline medium and pulse labeled with 35S-methionine incorporate more 35S-methionine into argininosuccinate synthetase protein than cells grown in arginine; the rate of disappearance of radioactively labeled enzyme is the same in citrulline- and arginine-grown cells. Arginine or a closely related metabolite thus appears to repress the synthesis of argininosuccinate synthetase of human lymphoblasts in culture.     

Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction

Argininosuccinate synthetase reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The structures of the enzyme from Thermus thermophilus HB8 complexed with intact ATP and substrates (citrulline and aspartate) and with AMP and product (argininosuccinate) have been determined at 2.1- and 2.0-A resolution, respectively. The enzyme does not show the ATP-induced domain rotation observed in the enzyme from Escherichia coli. In the enzyme-substrate complex, the reaction sites of ATP and the bound substrates are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level. The mobility of the triphosphate group in ATP and the side chain of citrulline play an important role in the catalytic action. The protonated amino group of the bound aspartate interacts with the alpha-phosphate of ATP and the ureido group of citrulline, thus stimulating the adenylation of citrulline. The enzyme-product complex explains how the citrullyl-AMP intermediate is bound to the active site. The stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS (argininosuccinate synthetase from T. thermophilus HB8) complexes.     

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.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

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.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Citrulline metabolism in normal and citrullinemic human lymphocyte lines

Citrullinemia is one of the five aminoacidurias associated with the Krebs-Henseleit urea cycle. A long-term lymphocyte line (UM-21) derived from a patient with this disease and nine of ten clones of this line were found to have no activity for the enzyme argininosuccinate synthetase (AS), as demonstrated by their inability to grow in medium in which citrulline had been substituted for arginine, by their inability to incorporate arginine-C14 derived from citrulline-C14 into cellular protein, and by direct enzyme assay. One clone had normal or nearly normal argininosuccinate synthetase activity, as demonstrated by the same criteria. Nutritional "variants" able to grow logarithmically in medium containing citrulline were isolated from UM-21 and three clones. The apparent Kms of AS for citrulline in UM-21, the ten clones, the variant lines, and a normal line were measured and fell into three groups: AS in UM-21 and nine clones had no measurable apparent Km for citrulline; AS in the variant cells had apparent Kms for citrulline of approximately 20 mM; and AS in the normal cell line and one clone had apparent Kms for citrulline of 0.2 mM. The data suggest that the defect in the citrullinemic cell lines is due to a mutation in the structural gene coding for argininosuccinate synthetase.     

Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity of Escherichia coli by Arginine Biosynthetic Precursors

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.     

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.     

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.     

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.     

Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.     

Control of Arginine Biosynthesis in Escherichia coli: Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity

In this study, we have extended our earlier observations indicating in vitro inhibition of arginyl-transfer ribonucleic acid synthetase (EC 6.1.13, arginine: soluble ribonucleic acid ligase, adenosine monophosphate) activity by the arginine biosynthetic precursors ornithine, citrulline, and argininosuccinate. Furthermore, we report evidence which suggest that this enzyme activity is inhibited by these arginine precursors in vivo and that this inhibition of activity results in a derepression of arginine biosynthesis.     

Substrate induced conformational changes in argininosuccinate synthetase.

Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. The crystal structures of Escherichia coli argininosuccinate synthetase (EAS) in complex with ATP and with ATP and citrulline have been determined at 2.0-A resolution. These are the first EAS structures to be solved in the presence of a nucleotide substrate and clearly identify the residues that interact with both ATP and citrulline. Two distinct conformations are revealed for ATP, both of which are believed to be catalytically relevant. In addition, comparisons of these EAS structures with those of the apoenzyme and EAS complexed with aspartate and citrulline (Lemke, C. T., and Howell, P. L. (2001) Structure (Lond.) 9, 1153-1164) provide structural evidence of ATP-induced conformational changes in the nucleotide binding domain. Combined, these structures also provide structural explanations of some of the observed kinetic properties of the enzyme and have enabled a detailed enzymatic mechanism of AS catalysis to be proposed.     

Crystal structure of argininosuccinate synthetase from Thermus thermophilus HB8. Structural basis for the catalytic action

Argininosuccinate synthetase catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its free form, complexed with intact ATP, and complexed with an ATP analogue (adenylyl imidodiphosphate) and substrate analogues (arginine and succinate) have been determined at 2.3-, 2.3-, and 1.95-A resolution, respectively. The structure is essentially the same as that of the Escherichia coli argininosuccinate synthetase. The small domain has the same fold as that of a new family of "N-type" ATP pyrophosphatases with the P-loop specific for the pyrophosphate of ATP. However, the enzyme shows the P-loop specific for the gamma-phosphate of ATP. The structure of the complex form is quite similar to that of the native one, indicating that no conformational change occurs upon the binding of ATP and the substrate analogues. ATP and the substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometrical orientation favorable to the catalytic action. The reaction mechanism so far proposed seems to be consistent with the locations of ATP and the substrate analogues. The reaction may proceed without the large conformational change of the enzyme proposed for the catalytic process.     

Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.     

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.