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)

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.
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PMID:[Role of the ribose residue of substrates in reactions catalyzed by ribonuclease]. 68 97

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
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PMID:Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis. 169 38

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.
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PMID:A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis. 271 39

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state.
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PMID:Active site dynamics of ribonuclease. 386 34

The terminal fragment obtained by hydrolysis with ribonuclease T(1) of the ribonucleic acid from the bacteriophage Qbeta has been isolated and purified. The results of chemical and enzymatic hydrolysis of this fragment and of the intact RNA itself indicate that the fragment has the composition (10 Cp, 4 Up), and that the RNA has the terminal sequence -GP(9 Cp, 4 Up) CpA. These conclusions are supported by the results of an application of the sedimentation-equilibrium method in which the molecular weight of the Qbeta fragment was compared with that of the corresponding fragment from f2 phage RNA for which the terminal sequence, -GpUpUpApCpCpApCpCpCpA had previously been determined.
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PMID:Comparison of RNA terminal sequences of phages f2 and Q-beta: chemical and sedimentation equilibrium studies. 565 59

A nucleolar ribonuclease specific for single-stranded ribonucleic acid (RNA) has been isolated and extensively purified from Ehrlich ascites carcinoma cells. The enzyme is optimally active at neutral pH and degrades RNA via a 2',3'-cyclic intermediate leaving 3'- or 2',3'-cyclic terminated oligonucleotides. The ribonuclease has an apparent molecular weight of 38 500 as judged by sedimentation equilibrium and is a basic protein having an isoelectric point greater than 9.0. The enzyme preferentially cleaves poly(C) over poly (U), poly(A), or poly(C).poly(I). Limit digestion products of poly(C) degratation are on the average tri-, tetra-, and pentanucleotides. In the partial digestion of yeast 5.8S rRNA, the nucleolar ribonuclease cleaves only CpA phosphodiester bonds. Spermidine, spermine, and histone I inhibit the activity of nucleolar ribonuclease. Antibodies directed toward pancreatic RNase do not cross-react with the Ehrlich nucleolar ribonuclease.
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PMID:Properties of a purified nucleolar ribonuclease from Ehrlich ascites carcinoma cells. 739 14

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

Phenylalanine120 is a candidate residue juxtaposing catalytic His12 and His119 in ribonuclease A (RNase A). To clarify its role in construction of the catalytic center, Phe120 was replaced by alanine, tryptophan, leucine, or glutamic acid by site-directed mutagenesis. The transphosphorylation and hydrolysis activities of the mutant RNase As, respectively, toward cytidinyl 3',5' adenosine (CpA) and cytidine 2',3' cyclic monophosphate (C>p) were compared with those of the wild type enzyme. The Km values of the two reactions increased markedly with slight changes in the Kcat values. The pKe values of His12 and His119 in the wild type and mutant enzymes, estimated from the pH dependence of the kcat/Km values, showed little change. The rate of carboxymethylation was reduced markedly by the mutations. The Ki values of the phosphate anion as to hydrolysis activity increased only slightly when Phe120 was replaced by leucine, tryptophan, or alanine. These findings suggest that Phe120 participates in the binding of the substrate, juxtaposing His12 and His119, and in stabilizing the transition state intermediate in the hydrolysis reaction. Furthermore, the decreases in the thermal denaturation temperatures of all the mutants, particularly F120E, indicate that Phe120 also helps maintain the conformational stability of RNase A.
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PMID:Role of Phe120 in the activity and structure of bovine pancreatic ribonuclease A. 968 34

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.
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PMID:Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity. 1033 57

Kinetic parameters of cleavage of CpA and UpA sequences in an oligoribonucleotide under the action of artificial ribonuclease ABL3C1 were measured. The compounds were built of RNA-binding domain B, catalytic fragment C, linker L3 comprising 3 methylene groups, and aliphatic fragment A. The rate of cleavage of phosphodiester bonds in CpA sequence within decaribonucleotide UUCAUGUAAA was shown to be 3.4 +/- 0.2 times higher than in UpA sequence. The rate of cleavage of phosphodiester bonds were found to depend on substrate length: a thousandfold increase in cleavage rate constant was observed for CpA sequence in decaribonucleotide as compared with diribonucleotide monophosphate CpA. A slight decrease in the cleavage rates was observed for the reactions proceeding in different buffers at pH 7.0: imidazole > HEPES > phosphate > cacodylate. At the same time, the ratio of cleavage rates for CpA and UpA sequences remained constant.
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PMID:[Kinetic parameters of hydrolysis of CpA and UpA sequences in an oligoribonucleotide by compounds functionally mimicking ribonuclease A]. 1250 May 46


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