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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Frequency-domain fluorescence resonance energy transfer and anisotropy measurements were performed to characterize conformational dynamics of an analog of the RNase S-peptide (residues 1-20). Trp was used as a donor by replacing Phe 8, and a dansyl acceptor group was introduced at position 1 or 18. The distance-distribution parameters, half width of the distribution, end-to-end diffusion coefficient, and to some extent anisotropy decays were sensitive to changes in the S-peptide conformation. The observed mean distance of about 13-14 A between residues 1 and 8 in the presence of 50% TFE and when bound to RNase S-protein is in reasonable accord with the X-ray structure of RNase. The mean distance of 9.3 A between residues 8 and 18 in the presence of 50% TFE is, however, significantly smaller than 15.3 A found for the S-protein complex. The half-width of the distance distribution increased from about 9 to 18 A for residues 1-8 and from about 6 to 14 A for segment 8-18 with the loss of helical structure. The half-widths of 9 A in the case of 1-8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 A half-width for segment 8-18 when it is random-coil is smaller than that expected for a random coil 11-residue segment. The donor-to-acceptor diffusion coefficients were less than 1 x 10(-7) cm2/s at 2 degrees C for both segments and increased to 1-2 x 10(-6) cm2/s at 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays. 824 Nov 20

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.
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PMID:[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. 825 Sep 78

The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution. The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity. Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances. Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes. The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands). However, some notable differences, which account for the enhanced thermostability of T. thermophilus RNase H, are observed in loop structures and side-chain conformations. The substitution of Gly for the left-handed helical residue (Lys95) in the E. coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom. Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.
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PMID:Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution. 838 28

Changes in biological properties of serum albumin, egg white lysozyme, human serum alpha-1 antiproteinase and human leukocyte ribonuclease in effect of interaction with the enzyme system composed of myeloperoxidase from human neutrophilic polymorphonuclear leukocytes, Cl- and H2O2 were investigated. All the studied proteins lost their biological functions and were denaturated, but the amounts of hydrogen peroxide necessary to produce these effects differed remarkably for each individual protein. The alpha-1 antiproteinase ability of binding to trypsin was abolished upon employing 1.2 mols of H2O2 per mol of alpha-1 antiproteinase. The lysozyme enzymatic activity was abolished when 1.4 mols of H2O2 per mol of lysozyme were employed. Albumin decreased its binding to specific antialbumin antibodies and entirely lost the binding properties when 2 mols and about 10 mols of H2O2 per mol of albumin were employed, respectively. On the other hand 18 mols of H2O2 per mol of human leukocyte ribonuclease were necessary to inactivate this enzyme. All the mentioned proteins were protected from losing their biological functions by excess of specific amino acids with affinity to hypochlorite: Alpha-1 antiproteinase by excess of N-acetylmethionine, lysozyme by N-acetylmethionine and N-acetyl glycyltryptophane, albumin by N-acetyl derivatives of methionine, cysteine, tryptophane and lysine, whereas ribonuclease was protected from denaturation by all above mentioned amino acid derivatives. None of the studied proteins was protected from denaturation by N-acetyl tyrosine, or phenylalanine.
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PMID:Inactivation and denaturation of some proteins by enzyme system: myeloperoxidase, chloride and hydrogen peroxide. 840 71

The distribution of neutral endopeptidase (NEP; EC 3.4.24.11) was examined in the alimentary tract of the rat. Immunoreactive NEP and NEP mRNA were localized to epithelial cells of the small intestine and to muscle cells in the stomach, small intestine, and colon by immunohistochemistry and in situ hybridization histochemistry. NEP antisera recognized a protein on Western blots of membranes from gastric, jejunal, and colonic mucosa and gastric muscle with an electrophoretic mobility identical to that of recombinant human NEP (approximately 95 kDa). An antisense cRNA probe to NEP hybridized to RNA of approximately 3.5 kb and approximately 6.5 kb, corresponding to the primary transcripts of rat NEP, on Northern blots of total RNA from the jejunal mucosa. NEP message was detected in mRNA from jejunal and colonic mucosa and gastric, jejunal, and colonic muscle using a ribonuclease protection assay. NEP enzymatic activity, assessed by DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was highest in homogenates of jejunal mucosa (868 +/- 98 pmol.h-1 x micrograms protein-1) and was between 49- and 413-fold lower in other gastrointestinal tissues. The cellular origin of NEP in the gastric and colonic mucosa could not be determined.
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PMID:Distribution and abundance of neutral endopeptidase (EC 3.4.24.11) in the alimentary tract of the rat. 846 Jul 3

RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.
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PMID:Structural determinants of the uridine-preferring specificity of RNase PL3. 870 17

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.
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PMID:1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin. 873 69

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.
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PMID:Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA. 891 3

Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala52 and Val74, resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu. All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified. Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains. A correlation was observed between the protein stability and the hydrophobicity of the substituted residue. As a result, a number of the mutant proteins were more stable than the wild-type protein. The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins. However, examination of the far- and near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein. These results suggest that the cavity in the hydrophobic core of E. coli RNase HI is conformationally fairly stable. This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein.
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PMID:Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core. 922 39

Barnase, a small extracellular ribonuclease from Bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly. Barnase has also evolved to be catalytically active. The active site of barnase and its binding site for barstar use the same subset of amino acids. The exception is Glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar. We examined in this work the contribution of Glu73 to both catalysis and barstar binding. Truncation mutants of the general base (Glu73 --> Ala or Ser) retain a residual RNase activity of about 0.3% while mutants with larger hydrophobic replacements (Glu 73 --> Trp or Phe) have virtually no catalytic activity. This, and binding data of 3'-GMP with the different barnase mutants suggest that the loss in activity results from the elimination of the general base, which can be substituted to some extent by water or other polar side-chains in truncation mutants. All of the Glu73 mutations lead to a weakening of the free energy of complex formation with barstar by 1.4 to 3.0 kcal/mol (including Gln). This is surprising, since Glu73 does not interact directly with barstar and there is an electrostatic repulsion between Glu73 on barnase and the negatively charged binding surface of barstar. A newly developed method of constructing double mutant cycles between multiple mutations at the same site appears to pinpoint a favourable interaction between Glu73 and one of its nearest neighbours in barstar, Asp39. The coupling energy between those residues is presumably indirect: the carboxylate of Glu73 organizes neighbouring positively charged groups in barnase, Lys27, Arg83, and Arg87 to interact with Asp39 in barstar. This emphasizes that an apparent interaction between a pair of residues as measured with double mutant cycles is the sum of their direct and indirect interactions.
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PMID:The role of Glu73 of barnase in catalysis and the binding of barstar. 923 5


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