Nelson Carvajal

Interaction of arginase with metal ions: studies of the enzyme from human liver and comparison with other arginases

As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.

Mutational analysis of substrate recognition by human arginase type I--agmatinase activity of the N130D variant.

Upon mutation of Asn130 to aspartate, the catalytic activity of human arginase I was reduced to ∼ 17% of wild‐type activity, the Km value for arginine was increased ∼ 9‐fold, and the kcat/Km value was reduced ∼ 50‐fold. The kinetic properties were much less affected by replacement of Asn130 with glutamine. In contrast with the wild‐type and N130Q enzymes, the N130D variant was active not only on arginine but also on its decarboxylated derivative, agmatine. Moreover, it exhibited no preferential substrate specificity for arginine over agmatine (kcat/Km values of 2.48 × 103 m−1·s−1 and 2.14 × 103 m−1·s−1, respectively). After dialysis against EDTA and assay in the absence of added Mn2+, the N130D mutant enzyme was inactive, whereas about 50% full activity was expressed by the wild‐type and N130Q variants. Mutations were not accompanied by changes in the tryptophan fluorescence properties, thermal stability or chromatographic behavior of the enzyme. An active site conformational change is proposed as an explanation for the altered substrate specificity and low catalytic efficiency of the N130D variant.

Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies. Alteration of substrate specificity by replacement of Asn149 with Asp.

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild‐type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The Km for arginine (1.4–1.6 mm) was not altered and the wild‐type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (Km = 2.5 ± 0.2 mm) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn2+ and contained < 0.1 Mn2+·subunit−1, whereas wild‐type and His145Asn enzymes were half active and contained 1.1 ± 0.1 Mn2+·subunit−1 and 1.3 ± 0.1 Mn2+·subunit−1, respectively. Manganese reactivation of metal‐free to half active species followed hyperbolic kinetics with Kd of 1.8 ± 0.2 × 10−8 m for the wild‐type and His145Asn enzymes and 16.2 ± 0.5 × 10−8 m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (λmax = 338–339 nm) and sensitivity to thermal inactivation were not altered. The Asn149→Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn2+A is the more tightly bound metal ion in arginase II.

Manganese-dependent inhibition of human liver arginase by borate.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus.

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.

Properties of arginase from the foot muscle of Chiton latus

Abstract 1. 1. Arginase activity was detected in the gill and foot muscle tissues of the marine mollusc Chiton latus (Polyplacophora). The muscle enzyme was partially purified and characterized. 2. 2. The enzyme has a mol. wt of about 79,000, which would correspond to a dimeric molecule. 3. 3. The K m values of arginine were 25 and 3 mM at pH 7.5 and 9.5, respectively. At pH 9.5, but not at pH 7.5, the enzyme was inhibited by high concentrations of arginine. 4. 4. The enzyme was inhibited by Zn 2+ and Ca 2+ and the enzyme inactivated by EDTA regained activity in the presence of Mn 2+ and to a much lower extent in the presence of Co 2+ , Ni 2+ and Ca 2+ . 5. 5. Significant inhibition was caused by ornithine, lysine and branched-chain amino acids. 6. 6. It is suggested that arginase might play a role in the adaptation of the mollusc to anoxic conditions.

Properties of arginase from the sea mollusc Concholepas concholepas

Abstract 1. 1. Arginase activity was detected in homogenates prepared from the gill of the sea mollusc Concholepas concholepas . From this tissue a partially purified preparation (sp. act. 30 units/mg of protein) was obtained and characterized. 2. 2. The metal ion requirement of the enzyme is satisfied by Mn 2+ , Co 2+ and Ni 2+ and a significant inhibition of the Mn 2+ -activated enzyme is caused by Cd 2+ , Mg 2+ and Zn 2+ . 3. 3. The enzyme exhibits Michaelis-Menten kinetics and at the pH optimum of 9.5 the K m for arginine was found to be 25 mM. Lysine and the product ornithine are competitive inhibitors while the inhibition caused by branched chain amino acids and proline is non-competitive. 4. 4. The enzyme has a molecular weight of about 27,500, which is in the order of the molecular weight of the subunits of oligomeric arginases. The molecular weight of the enzyme from Concholepas concholepas is not altered by addition or withdrawal of metal ions.

Expression and localization of an agmatinase-like protein in the rat brain

Agmatinase catalyzes the hydrolysis of agmatine into putrescine and urea, and agmatine (decarboxylated l-arginine) plays several roles in mammalian tissues, including neurotransmitter/neuromodulatory actions in the brain. Injection of agmatine in animals produces anticonvulsant, antineurotoxic and antidepressant-like actions. Information regarding the enzymatic aspects of agmatine metabolism in mammals, especially related to its degradation, is relatively scarce. The explanation for this is the lack of enzymatically active preparations of mammalian agmatinase. Recently, we have cloned a protein from a cDNA rat brain library having agmatinase activity although its amino acid sequence greatly differs from all known agmatinases, we called agmatinase-like protein. In this work, we analyzed the expression of this enzyme in the rat brain by means of RT-PCR and immunohistochemical analysis using a polyclonal antibody generated against the recombinant agmatinase-like protein. The agmatinase-like protein was detected in the hypothalamus in glial cells and arcuate nucleus neurons, and in hippocampus astrocytes and neurons, but not in brain cortex. In general, detected localization of agmatinase-like protein coincides with that described for its substrate agmatine and our results help to explain several reported effects of agmatine in the brain. Concretely, a role in the regulation of intracellular concentrations of the neurotransmitter/neuromodulator agmatine is suggested for the brain agmatinase-like protein.

Subcellular localization, metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Abstract Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn2+ followed hyperbolic kinetics with a KMn value of 0.10 ± 0.02 μM. In addition to Mn2+, the metal ion requirement of the enzyme was satisfied by Ni2+, Cd2+ and Co2+; Zn2+ was inhibitory to ail the Values of Km for arginine and Ki for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; Km values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.

Studies on the functional significance of a C-terminal S-shaped motif in human arginase type I: essentiality for cooperative effects.

The functional significance of a C-terminal S-shaped motif (residues 304-322) in human arginase I was explored by examining the kinetic properties of the R308A mutant and truncated species terminating in either Arg-308 or Ala-308. Replacement of Arg-308 with alanine, with or without truncation, yielded monomeric species. All mutants were kinetically indistinguishable from the wild-type enzyme at the optimum pH of 9.5. At the more physiological, pH 7.5, hyperbolic kinetics was observed for all the mutants, in contrast with the cooperative behavior exhibited by the wild-type species. In the presence of 2mM guanidinium chloride (Gdn(+)), the single mutant R308A changed to a trimeric and kinetically cooperative form, whereas the other enzyme variants were not altered. The S-shaped motif is suggested as essential for the cooperative response of the enzyme to l-arginine at pH 7.5. Gdn(+) is suggested to mimic the guanidine group of Arg-308 at the monomer-monomer interface.