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)

The replacement of genetically deficient enzymes in patients with inherited metabolic disorders by infusion of purified enzymes or by organ transplantation has had very limited success, although good results with bone marrow transplantation have been obtained in some patients with mucopolysaccharidosis, Gaucher disease and inherited immunodeficiency diseases. Genetic engineering of the patient's lymphocytes may ultimately render these approaches redundant, at least for some of these diseases. Treatment of chronic pancreatic insufficiency and of disaccharidase deficiency with oral enzymes can be very effective; therapy can be monitored in the latter by measuring the breath hydrogen excretion and in the former by a range of tests of which stool chymotrypsin assay is the most convenient. Treatment of acute myocardial infarction by intracoronary perfusion of thrombolytic enzymes can improve both cardiac function and long-term survival if given early enough. Successful reperfusion can be identified by changes in the kinetics of serum enzyme release and clearance, especially for the isoenzymes and isoforms of creatine kinase. In cancer chemotherapy, L-asparaginase has long been a useful adjunct in the treatment of acute lymphoblastic leukemia, but recent experience suggests a role in acute nonlymphoblastic leukemia as well.
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PMID:Enzymes as agents for the treatment of disease. 157 79

The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily determined at room temperature in solution by monitoring the triplet absorption or emission following an exciting laser pulse. The dioxygen triplet quenching constants, can be measured for all these molecules and compared to the analogous singlet values determined by fluorescence methods. The dioxygen triplet quenching constant (tkq) ranged from a high of 5.1.10(9) M-1.s-1 for the exposed indole of corticotropin to a low of 0.1.10(9) M-1.s-1 for the buried indole of asparaginase. The ratio of these values with their respective dioxygen singlet quenching constants (skq), tkq/skq, ranged from 0.3 to 0.6 for aqueous exposed polypeptide indoles. For globular proteins the tkq/skq value is observed to be 0.2 +/- 0.1. This lower value for protein indoles is not attributable to 'bulk' environmental or hydrogen bonding effects, since the magnitude of tkq/skq (= 0.5 +/- 0.1) for model indoles was independent of solvent dielectric constant, polarity, and proticity. Temperature-dependence studies were done to test whether tkq could be used to characterize the nature of the protein matrix. The activation energy (Ea) for tkq was found to be 11 +/- 2 kcal/mol for most proteins. This Ea was independent of whether the indole side-chain was solvent exposed or buried in the non-aqueous protein interior. Large Ea values were also obtained for model indoles, naphthalene and nalidixic acid, dissolved in water, whereas the same compounds dissolved in 95% ethanol exhibited much smaller Ea values. These data, in combination with the observation that the tkq of model indoles is insensitive to changes in solvent viscosity, indicate that dioxygen quenching at the triplet level can not be easily used to characterize the dynamics of proteins.
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PMID:Determination of the dioxygen quenching constant for protein and model indole triplets. 319 Nov 39

An activated magnetic modifier, which could render biological materials magnetic property, was synthesized in following two steps: oxidation of ferrous ions (Fe2+) with hydrogen peroxide in the presence of alpha, omega-dicarboxymethylpoly(oxyethylene) (DCPEG) to obtain DCPEG-magnetite (Fe3O4); free carboxyl groups in the DCPEG-magnetite were activated with N-hydroxysuccinimide. By coupling the activated magnetic modifier to amino groups of lipase or L-asparaginase, magnetic enzymes were prepared. They dispersed stably not only in aqueous solution but also in organic solvents with high enzymic activities. Magnetic enzymes were readily recovered from reaction mixture in a magnetic field of 6000 Oe without loss of enzymic activity.
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PMID:Chemical modification of enzymes with activated magnetic modifier. 359 78

Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
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PMID:Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F. 749 89

Parameters of fluorescence of three single-tryptophan-containing proteins and of two log-normal components of proteinase K (2 tryptophans) were analyzed in relation to the microenvironment characteristics of indolic atoms in crystal structures of the proteins. For this purpose, it was constructed a system of microenvironment description including accessibility of the atoms to the bulk and bound water; the density, polarity and mobility of environment within radii of 5.5 and 7.5 A from each indolic atom; and the existence of eventual partners in hydrogen bonding with excited fluorophore. The analysis showed that, in the cases of the most shorter-wavelength emission bands (those structured at 308 nm for azurin and at 316 nm for L-asparaginase), as well as of the monomer melittin band at 350 nm, the microenvironment characteristics well agreed to those predicted in the model of discrete states of tryptophan in proteins [1,3,7] and can be used for assignment of protein fluorescence spectral components to individual tryptophan residues. However, differences of the microenvironment parameters included in the system are little discernible for the component bands of proteinase K emission at ca. 330 and 340 nm. In order to reliably assign such components of tryptophan fluorescence, it seems to be sufficient to take into account some additional structural characteristics, which could be revealed in a comprehensive analysis of a great number of proteins possessing such spectral components.
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PMID:[Assignment of a component of protein fluorescence spectra to tryptophan residues by their three-dimensional microoenvironmental properties]. 917 73

A conservative and apparently harmless A176V mutation in intracellular S. cerevisiae L-asparaginase (ScerAI) completely abolishes the enzyme activity. Sequence and structural comparisons with type II bacterial L-asparaginases show that the mutated residue is in a very conservative region and plays a vital role in the cohesion of functional tetramers of these enzymes through participation in side-chain...main-chain (Ser) Oy...O (Ala) hydrogen bonds across the tetramer interface. The fact that bacterial L-asparaginases of type I show less conservation in this region suggests that they may have different quaternary structure while adopting the subunit fold and intimate dimer architecture of type II enzymes. A comparison of all available sequences of microbial L-asparaginases confirms that separate intra- and extra-cellular enzymes evolved in prokaryotes and eukaryotes independently. However, an analysis of the available complete genome sequences reveals a surprising fact that Haemophilus influenzae possesses only a type II asparaginase while the archaebacterium Methanococcus jannaschii has a type I gene, but not a type II.
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PMID:Why a "benign" mutation kills enzyme activity. Structure-based analysis of the A176V mutant of Saccharomyces cerevisiae L-asparaginase I. 951 60

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism.
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PMID:Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes. 1070 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

AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell.
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PMID:Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L-asparaginase I. 1745 45

A novel assay for the determination of l-asparaginase activity in human plasma is described that is based on the HPLC quantitation of l-aspartic acid produced during enzyme incubation. Methods for monitoring l-asparagine depletion are also described. Chromatography of l-aspartic acid, l-asparagine and l-homoserine (the internal standard) involved derivatization with o-pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C(18) column using a 1 mL/min flow rate and a mobile phase consisting of di-potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l-aspartic acid, l-asparagine and l-homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l-asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l-asparaginase as treatment for acute lymphoblastic leukaemia.
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PMID:An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. colil-asparaginase for the treatment of acute lymphoblastic leukaemia. 1882 71


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