L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

Structural and functional insights into Erwinia carotovora L-asparaginase

Bacterial L-asparaginases are enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral-asparaginase in the presence of L-Asp was determined at 2.5 A resolution and refined to an R cryst of 19.2 (R free = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.     

Sulfhydryl groups of L-asparaginase A from Pseudomonas fluorescens AG]

It has been demonstrated that the activity of asparaginase A from Ps. fluorescens AG is completely inhibited by 10(-4) M p-chloromercurybenzoate and by 70-85% by Zn2+, Ca2+ and Cu2+ (2.10(-2) M). Iodoacetate, iodoacetamide, N-ethylimide of maleic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) do not decrease the enzyme activity. Dithiothreitol and beta-mercaptoethanol reactivate the enzyme. L-asparagine, the substrate of asparaginase, protects the enzyme in a large degree against the inhibitory action of p-chloromercurybenzoate. p-chloromercurybenzoate induces a sharp increase in the asparaginase inactivation rate at acidic (6.5--5.5) and alkaline (7.5-8.5) values of pH. The enzyme modification by p-chloromercurybenzoate does not change the Km value for L-asparagine, but decreases Vmax. Thus it may be assumed, that asparaginase from Ps. fluorescens AG contains sulfhydryl groups essential for the enzyme activity.     

Asparagine utilization in Escherichia coli

Asparagine-requiring auxotrophs of Escherichia coli K-12 that have an active cytoplasmic asparaginase do not conserve asparagine supplements for use in protein synthesis. Asparagine molecules entering the cell in excess of the pool required for use of this amino acid in protein synthesis are rapidly degraded rather than accumulated. Supplements are conserved when asparagine degradation is inhibited by the asparagine analogue 5-diazo-4-oxo-l-norvaline (DONV) or mutation to cytoplasmic asparaginase deficiency. A strain deficient in cytoplasmic asparaginase required approximately 260 mumol of asparagine for the synthesis of 1 g of cellular protein. The cytoplasmic asparaginase (asparaginase I) is required for growth of cells when asparagine is the nitrogen source. This enzyme has an apparent K(m) for l-asparagine of 3.5 mM, and asparaginase activity is competitively inhibited by DONV with an apparent K(i) of 2 mM. The analogue provides a time-dependent, irreversible inhibition of cytoplasmic asparaginase activity in the absence of asparagine.     

Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.     

Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

Tumor inhibitory and non-tumor inhibitory L-asparaginases from Pseudomonas geniculata.

Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.     

Activation and stabilization of asparaginase by anti-asparaginase IgG and its Fab

Modified asparaginase, in which 4 tryptophan residues were modified with 2-hydroxy-5-nitrobenzyl bromide, had little enzymic activity and retained immunoreactivity [(1976) FEBS Lett. 65, 11-15]. Addition of IgG or its Fab towards asparaginase to the modified asparaginase gave rise to marked enhancement of the enzymic activity. Native asparaginase (4 subunits) lost the enzymic activity due to dissociation into subunits by dilution of the enzyme solution. However, in the presence of Fab, asparaginase did not lose enzymic activity on dilution, probably due to no dissociation into subunits occurring.     

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Reversible self-phosphorylation of asparaginase complex in Leptosphaeria michotii: characterization of associated protein kinase and protein phosphatase activities

Regulation of the asparaginase activity rhythm in L. michotii has previously been shown to be dependent on a reversible phosphorylation process. Asparaginase was isolated as a purified protein complex having self-phosphorylating capacities, which were analyzed. In vivo phosphorylation of asparaginase complex was performed synchronously with the rhythm of asparaginase activity. In vitro self-phosphorylation of asparaginase complex resulted from the activity of an ATP-Mg2+-dependent protein kinase, which phosphorylated protein at threonine residues and was not dependent on cyclic AMP, Ca2+ or calmodulin. Dephosphorylation of this complex was due to a Mg2+-Zn2+-dependent protein phosphatase, molybdate inhibited, the specificity of which, for low-molecular-weight nonprotein phosphoesters, was broad.     

L-asparaginase from developing seeds of Lupinus arboreus.

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.     

L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase.

An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.     

Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of enzyme. Methoxy-p-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of the pegylation procedure resulted in high level of enzyme modification. Under 4.5 molar excess of the modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for production of asparaginase with improved therapeutic properties.     

Proteolysis and the diurnal decrease of asparaginase activity

The mechanism responsible for the decrease in asparaginase (EC 3.5.1.1) activity in darkened leaves of Pisum sativum L. cv. Little Marvel was investigated. Asparaginase activity, obtained from half-expanded leaves harvested at the end of the dark period, or during the light periods, was inactivated by bromelain (EC 3.4.22.4), ficin (EC 3.4.22.3), both thiol proteases, and trypsin (EC 3.4.21.4), a serine protease. Thrombin (EC 3.4.21.5), pepsin (EC 3.4.23.1), or carboxypeptidase A (EC 3.4.17.1) had no effect on dark- or light-harvested asparaginase preparations. Inactivation of asparaginase activity in crude or purified preparations by ficin was not observed in the presence of leupeptin (an inhibitor of thiol proteases). Supplying leupeptin to detached half-expanded leaves had no effect on the increase of asparaginase observed at the start of the light period, while it maintained asparaginase activity at high levels in leaves excised during or at the end of the light period. These results suggest that decreased asparaginase activity in vivo is brought about by thiol-dependent proteases.     

Extracellular Asparaginase from Candida utilis,Its Properties as Glycoprotein and Antitumor Activities

Purified Candida asparaginase was proved to be homogeneous by gel filtration, ultra-centrifugation and disc electrophoresis. The enzyme was found to have properties as glycoprotein containing mannose. The ratio of mannose to protein was 1 to 2 in purified enzyme. Specific activity was 5500 units per nag of protein. Isoelectric point was pH 4 to 4.5 and sedimentation coefficient was found to be about 8.2 S. Antitumor activity of Candida asparaginase was inferior to E. coli enzyme. It was thought as the reason why the Candida asparaginase had less affinity to l-asparagine and it was cleared faster from the blood than E. coli asparaginase.     

Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.