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)

In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.
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PMID:Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate. 894 69

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

To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcoreTM). The kon and koff values for the binding of the enzyme to the 36-bp substrate were 1.5 x 10(6) M-1 s-1 and 3.2 x 10(-2) s-1, respectively. Similar values were obtained with the shorter substrates. Using uncleavable 2'-O-methylated RNA-DNA substrates, values for kon and koff were 2.1 x 10(5) M-1 s-1 and 1.3 x 10(-1) s-1 in the absence of Mg2+ that were further reduced in the presence of Mg2+ to 7.4 x 10(3) M-1 s-1 and 2.6 x 10(-2) s-1. Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp134 replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate. Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9-10 bp of RNA-DNA hybrid.
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PMID:Kinetic and stoichiometric analysis for the binding of Escherichia coli ribonuclease HI to RNA-DNA hybrids using surface plasmon resonance. 926 40

The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam'. The chimeric Pam residue integrates the heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position. The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme structure/ function relationships in a native-like peptidyl architecture.
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PMID:Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics. 927 83

Restrictocin is a small basic protein produced by the fungus Aspergillus restrictus. It potently inhibits protein synthesis in eukaryotic cells by specifically cleaving a single phosphodiester bond in 28S rRNA. A histidine residue at position 49 in restrictocin has been implicated in its active site. A mutant of restrictocin in which the histidine at position 49 was changed to an alanine was constructed by site-directed mutagenesis, and the protein was expressed in Escherichia coli. The mutant and the wild type proteins were found to be structurally identical. Unlike restrictocin, the restrictocin H49A mutant did not cleave the ribosomal RNA specifically at the target phosphodiester bond; instead, it extensively degraded the RNA substrate with altered specificity. The mutant exhibited a high ribonuclease activity compared to restrictocin on yeast tRNA, and poly(U) and poly(C). The mutant also poorly inhibited protein synthesis in eukaryotic cells as well as in a cell free system. We therefore propose that histidine 49 of restrictocin is not involved per se in the enzymatic activity; however, it does play a crucial role in the specific recognition of the target sequence by restrictocin.
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PMID:A single amino acid substitution in ribonucleolytic toxin restrictocin abolishes its specific substrate recognition activity. 935 40

Galectin-3 is a beta-galactoside-specific lectin that is a pre-mRNA splicing factor. Here we report the genomic organization of the human LGALS3 (galectin-3) gene and functional characterization of the promoter. Southern blot analysis of genomic DNA revealed that galectin-3 is coded by a single gene in the human genome. The gene is composed of six exons and five introns, spanning a total of approximately 17 kilobases (kb). Based on primer extension and ribonuclease protection analyses, there are two transcription initiation sites located 52 and 50 nucleotides (nt) upstream of the exon I-intron 1 border, and defined here as +1a and +1b, respectively. The translation start site is in exon II. The ribonucleoprotein-like N-terminal domain, containing the proline-glycine-alanine-tyrosine (PGAY) repeat motif, is found entirely within exon III. The carbohydrate recognition sequence is found entirely within exon V. Genomic fragments encompassing -836 to +141 nt (relative to +1a) have significant promoter activity when linked to the luciferase reporter gene and transiently transfected into HeLa cells or human diploid fibroblasts. Quiescent fibroblasts have low promoter activity but the activity increases 100-fold following serum addition. Serum responsive activation regions in the promoter are located between -513 and -339 nt and between -339 and -229 nt; an additional activation region may be located between -105 and -15 nt. Because galectin-3 is an immediate-early gene whose expression is dependent on the proliferative state of the cell, this study provides the basis for determining the molecular mechanisms of transcriptional regulation in neoplasia or cellular senescence.
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PMID:The human LGALS3 (galectin-3) gene: determination of the gene structure and functional characterization of the promoter. 943 77

The relationship between the structural stability and the internal motions of proteins was investigated through measurements of 15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His), which has a higher melting temperature by 20.2 degreesC. For most of the residues, the generalized order parameters (S2) obtained from 15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast H-D exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions. Several local mutational effects were observed for residues close to the mutation sites as follows. The S2 value significantly increased for Lys96 and Val98, which indicated that motions on the pico- to nanosecond time-scale became restricted within a protruding region including the Lys95-->Gly mutation site. In contrast, slight decreases in S2, and drastic increases in the chemical exchange motion on the micro- to millisecond time-scale (Deltaex), were observed for residues located in the joining region between the protrusion and the major domain of the protein. These changes may be caused by the elimination of the bulky Lys95 side-chain at the center of the protrusion. Deltaex observed for residues in alpha-helix I of the wild-type protein was reduced for the mutant, probably because a cavity in the hydrophobic core is filled by the Val74-->Leu mutation. The local breathing at position 134 was restricted by the Asp134-->His mutation, probably because the reduction of the negative charge repulsion contributes to the stability of the native major conformation relative to the breathing conformations around position 134.
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PMID:Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses. 953 89

Human angiogenin (Ang), a homologue of bovine pancreatic ribonuclease A (RNase A), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of RNase A but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of RNase A and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of RNase A. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the RNase A loop 65-72 from Ang has been investigated by reexamining the earlier derivative ARH-I (in which Ang residues 58-70 have been replaced by residues 59-73 of RNase) and generating new derivatives of this hybrid protein. The results suggest that the RNase A segment of ARH-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.
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PMID:Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123. 957 71

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.
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PMID:Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase). 964 74


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