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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the difference decreased when the number of contacts per nonpolar group and/or atom were calculated. Three main nuclei and five microclusters were identified, and their quantitative parameters were calculated. These nuclei include hydrophobic residues with a substantial number of nonpolar contacts with the environment (Phe 8, Phe 120, Phe 46, Tyr 25, Tyr 97, Ile 107, Leu 35, Ile 81, Val 54, Val 108, Met 29, Met 30). Hydrophobic nuclei of RNase A differ in shape and in composition, in the number of intranuclear contacts and of associated residues, as well as in their internal mobility. All eight cysteine residues are involved in nonpolar interactions with amino acid residues of hydrophobic nuclei. Active site amino acid residues of RNase A form a noncovalent contact network comprised of themselves, as well as of many conserved residues from hydrophobic nuclei. Sequence alignment with some other members of the RNase A family of proteins shows remarkable similarity in positions and in conservation of the main nonpolar residues, comprising cores of two (out of three) hydrophobic nuclei. A correlation was shown to exist between the average density of contacts for side-chain atoms and the number of amino acids to be found in the appropriate positions in the sequences of related mammalian ribonucleases. However, there are certain amino acid positions in the third, smaller nucleus, which are highly variable within the family. Taking into account that this nucleus is composed of residues belonging to different elements of the secondary structure, it is likely that the mutual orientation of these elements can be somehow different for these proteins.
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PMID:Intramolecular interactions in pancreatic ribonucleases. 130 82

Non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant (3)JHNHalpha and integration of the nuclear Overhauser HN-Halpha cross peak, positive theta-angles could be determined reliably to 60 degrees +/- 30 degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive theta-angles can also occur in non-glycine residues and in the four proteins, positive theta-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured (3)JHNHalpha coupling constants and the intensity of the intraresidue HN-Halpha NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wuthrich, K. (1984) J. Mol. Biol., 180, 741-751; Ludvigsen, S. Andersen, K.V. and Poulsen, F.M. (1991) J Mol. Biol., 217, 731-736).
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PMID:Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy. 139 67

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.
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PMID:Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function. 142 2

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.
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PMID:Effect of active site residues in barnase on activity and stability. 160 71

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.
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PMID:Heat capacity changes for protein-peptide interactions in the ribonuclease S system. 173 99

The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.
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PMID:Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia. 173 52

A class of cytosolic proteins has been identified that are degraded faster (have shorter half-lives) in human diploid fibroblasts deprived of serum. In RNase A, a model protein used for these studies, a pentapeptide comprising amino acids 7-11, Lys-Phe-Glu-Arg-Gln or KFERQ, is responsible for its enhanced degradation. The cytosolic proteins that are degraded faster during serum deprivation are recognized by an antiKFERQ antibody and, therefore, probably contain variations of the KFERQ motif. These cytosolic proteins are degraded in lysosomes. Transport into lysosomes in vitro is stimulated by ATP and the heat shock cognate protein of 73 kDa (hsc73).
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PMID:Targeting specific proteins for lysosomal proteolysis. 180 3

The primary structure of nuclease P1, which cleaves both RNA and single-stranded DNA, from Penicillium citrinum was elucidated. The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter protease I of the reduced and S-aminoethylated protein and by digestion with Staphylococcus aureus V8 protease of the reduced and S-carboxymethylated protein. Four half-cystine residues were assigned to Cys72-Cys217 and Cys80-Cys85. N-Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197. Fast-atom-bombardment and laser-ionization MS were successfully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively. Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease S1 from Aspergillus oryzae. The His-Phe-Xaa-Asp-Ala sequence (positions 60-64) is similar to the sequence (His-Phe-Asp-Ala) involving the active-site His119 of bovine pancreatic RNase A, and the Pro-Leu-His sequence (positions 124-126) is identical with the sequence involving the active-site His134 of porcine pancreatic DNase I.
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PMID:Primary structure of nuclease P1 from Penicillium citrinum. 191 39


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