Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.1 (asparaginase)
2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Asparagine synthetase B (AS) is the primary enzyme responsible for asparagine synthesis in plants. Routine biochemical studies of this enzyme's activity have been hindered by several problems including enzyme instability and rapid physiological turnover, endogenous inhibitors, competing pathways, and asparaginase activity. We describe an extraction procedure and assay conditions that provide a reliable, direct assay for the determination of AS activity in crude plant extracts. This assay performed well with several leguminous species and the enzyme preparation retained activity for up to 3 weeks when stored at -80 degrees C. Radio-HPLC detection enabled quantitative measurement of de novo aspargine synthesis in the extracts. Optimal activity was obtained with 1 mM glutamine and 10 mM ATP in the reaction assay. Aminooxyacetic acid (AOA, 1 mM) which prevents the assimilation of aspartate into the TCA cycle, was necessary to measure AS activity in peas, but not in lupine or soybean.
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PMID:Measuring asparagine synthetase activity in crude plant extracts. 1082 80

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.
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PMID:Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. 1110 75

L-asparaginase (EC 3.5.1.1) was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase was found to be 33 kDa by SDS-PAGE, whereas by Sephacryl S-300 superfine column it was found to be 200 kDa, indicating that the enzyme in the native stage acts as hexamer. It is a thermostable enzyme and keeps all of its activity at 80 degrees C for 10 min. The antiproliferative activity of the purified L-asparaginase from T. thermiphilus was tested against the following human cell lines: K-562 (chronic myelogenous leukemia), Raji (Burkitt's lymphoma), SK-N-MC (primitive neuroectodermal tumor), HeLa (cervical cancer), BT20 and MCF7 (breast cancers), HT-29 (human colon cancer), and OAW-42 (ovarian cancer). The antiproliferative activity of T. thermophilus enzyme was compared with Erwinase, the commercially available L-asparaginase from Erwinia corotovora. The potency difference between the two L-asparaginases was greater in HeLa and SK-N-MC than in other cell lines. The fact that L-asparaginase from T. thermophilus does not hydrolyse L-glutamine makes it advantageous for future clinical trials.
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PMID:Antitumor activity of L-asparaginase from Thermus thermophilus. 1126 87

Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide. In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc). Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes. The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode. The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side. The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15. Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site. Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value. A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity.
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PMID:Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. 1134 30

Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12 h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine.
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PMID:Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells. 1148 52

Cell shrinkage and loss of cell viability by apoptosis have been examined in cultured CD95(Fas/Apo-1)-expressing leukemia-derived CEM and HL-60 cells subjected to acute deprivation of glutamine, a major compatible osmolyte engaged in cell volume control. Glutamine deprivation-mediated cell shrinkage promoted a ligand-independent activation of the CD95-mediated apoptotic pathway. Cell transfection with plasmids expressing FADD-DN or v-Flip viral proteins pointed to a functional clustering of CD95 receptors at the cell surface with activation of the 'extrinsic pathway' caspase cascade. Accordingly, cell shrinkage did not induce apoptosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with surrogate compatible osmolytes counteracted cell volume decrement and protected the CD95-expressing cells from apoptosis. A glutamine deprivation-dependent cell shrinkage with activation of the CD95-mediated pathway was also observed when asparaginase was added to the medium. Asparagine depletion had no role in this process. The cell-size shrinkage-dependent apoptosis induced by glutamine restriction in CD95-expressing leukemic cells may therefore be of clinical relevance in amidohydrolase enzyme therapies.
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PMID:Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis. 1159 98

For this study, 118 children with standard-risk acute lymphoblastic leukemia (ALL) were given randomized assignments to receive native or pegylated Escherichia coli asparaginase as part of induction and 2 delayed intensification phases. Patients treated with pegaspargase had more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow aspirates and more prolonged asparaginase activity than those treated with native asparaginase. In the first delayed intensification phase, 26% of native asparaginase patients had high-titer antibodies, whereas 2% of pegaspargase patients had those levels. High-titer antibodies were associated with low asparaginase activity in the native arm, but not in the pegaspargase arm. Adverse events, infections, and hospitalization were similar between arms. Event-free survival at 3 years was 82%. A population pharmacodynamic model using the nonlinear mixed effects model (NONMEM) program was developed that closely fit the measured enzyme activity and asparagine concentrations. Half-lives of asparaginase were 5.5 days and 26 hours for pegaspargase and native asparaginase, respectively. There was correlation between asparaginase enzymatic activity and depletion of asparagine or glutamine in serum. In cerebrospinal fluid asparagine, depletion was similar with both enzyme preparations. Intensive pegaspargase for newly diagnosed ALL should be tested further in a larger population.
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PMID:A randomized comparison of native Escherichia coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children's Cancer Group study. 1221 Nov 94

Asparaginase production by a mesophilic strain of Erwinia sp. was examined; the maximum of activity was found at 40 degrees C and pH 8.5. Among the various carbon sources, mannitol was shown to be the best for production of activity. Inorganic nitrogen sources were better than the organic ones. The enzyme activity was not inhibited by 10 mmol/L metal ions (Na+, K+, Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Zn2+); the activity was strongly inhibited by addition of EDTA. L-Arginine, DL-alanine, L-asparagine and L-glutamine stimulated the L-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively. The combination of L-arginine, L-asparagine and L-glutamine synergistically stimulated the asparaginase up to 5.8 fold.
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PMID:Production and properties of asparaginase from a new Erwinia sp. 1250 89

L-asparaginase production was investigated in the filamentous fungi Aspergillus tamarii and Aspergillus terreus. The fungi were cultivated in medium containing different nitrogen sources. A. terreus showed the highest L-asparaginase (activity) production level (58 U/L) when cultivated in a 2% proline medium. Both fungi presented the lowest level of L-asparaginase production in the presence of glutamine and urea as nitrogen sources. These results suggest that L-asparaginase production by of filamentous fungi is under nitrogen regulation.
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PMID:Production of L-asparaginase by filamentous fungi. 1554 11

Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA(Gln) and the other subunit, GatE. The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA(Gln) accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from gamma-phosphoryl-Glu-tRNA(Gln), the proposed high energy intermediate in Glu-tRNA(Gln) transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA(Gln) kinase. The activation of Glu-tRNA(Gln) via gamma-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.
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PMID:Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase. 1561 Nov 11


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