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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The X-ray structures of two complexes of bovine ribonuclease-A produced by soaking pre-grown crystals in solutions of the inhibitors cytidylyl-2',5'-guanosine (2',5' CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG) have been determined at 1.5 A resolution and refined by restrained least squares to R = 21.0% for 17,855 reflections, and R = 19.1% for 16,347 reflections, respectively. Binding of the substrate analogs to the protein has taken place in a completely unexpected and previously unreported manner. In each case the guanine base occupies the well characterized B1 pyrimidine binding site adjacent to Thr-45 (described by Richards, F.M., Wyckoff, H.W., Carlson, W.D., Allewell, N.M., Lee, B. and Mitsui, Y. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 35-54, and others including Palmer, R.A., Moss, D.S., Haneef, I. and Borkakoti, N. (1984) Biochim. Biophys. Acta 785, 81-88) having entered through a secondary channel external to the active site itself. We designate this reversed non-productive mode as retro-binding. In this mode of binding the SO4(2-) anion bound in the active site of the native protein crystals (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217) has not been displaced by the phosphate of the inhibitor molecule as originally anticipated and observed in other studies. Instead the CMP or dCMP moiety of the inhibitor molecule is held loosely in a channel running towards the surface of the protein molecule and is thus completely external to the active site. Consequently, although it has been possible to model them, no attempt has been made to refine either the disordered cytosine in the CpG complex or the deoxycytosine in the dCpdG complex. The traditional B2 purine binding site of RNase (Richards et al., 1971) is unoccupied by the soaked inhibitors. Important changes that have taken place in the protein structure include: stabilization of both Lys-41 and Gln-11 via H-bonding to SO4(2-); stabilization of His-119 in the A conformation (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217); and stabilization of SO4(2-) by H-bonds formed with the retro-bound guanine base. Binding of the inhibitors and stabilization of the active site is accompanied by displacement and redistribution of solvent molecules.
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PMID:Novel non-productively bound ribonuclease inhibitor complexes--high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2',5'-guanosine (2',5'CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG). 176 78

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.
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PMID:Purification and characterization of two ribonucleases from developing tomato fruit. 192 99

Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.
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PMID:Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR. 195 71

The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.
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PMID:Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution. 202 57

The cell-envelope antigens of Peptostreptococcus anaerobius were extracted from intact cells by autoclave or alkaline treatment. The purified species-specific antigen (G) was identified among several polysaccharides obtained from the extracts by successive treatments with ribonuclease and pronase followed by ion-exchange and gel-filtration chromatography. G was investigated by 13C- and 31P-n.m.r. spectroscopy, titrimetry, elemental analysis, and gas-liquid chromatography. Oxidation of G with NaIO4 followed by reduction with NaBH4 and mild acid hydrolysis yielded the Smith degradation product of G (GS). Treatment of G and GS with 48% HF gave the respective dephosphorylated products GF and GSF. The structures of GS, GF, and GSF were investigated by 13C-n.m.r. spectroscopy, methylation analysis, and gas-liquid chromatography-mass spectrometry. The principal constituents of G were 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), D-glyceric acid, and phosphate as a diester, in the ratio 2:1:1, and a minor amount of D-glucose (beta-D-Glcp). GS contained D-GlcNAc, D-glyceric acid, glycerol, and phosphate in a 1:1:1:1 ratio. GF and GSF contained D-GlcNAc and D-glyceric acid in the ratios 2:1 and 1:1, respectively. A structure for the principal repeating unit of polymeric G compatible with the analytical data consists of alpha-D-GlcpNAc-(1----3)-alpha-D-GlcpNAc-(1----2)-D-glyceric acid units linked through C-6'-C-6" phosphate diester bridges. This structure is novel for two reasons: (a) unsubstituted glyceric acid residues occur as aglycons in the repeating structure, and (b) phosphate diester bridges link nonanomeric glycose carbons in a non-nucleic acid polymer. The structural role of the minor amount of beta-D-Glcp in G remains unknown.
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PMID:Polysaccharides from Peptostreptococcus anaerobius and structure of the species-specific antigen. 207 10

The absorbance peak in the near ultraviolet electron-transfer spectrum of the oxyvanadium constellation in the "transition-state-analogue complexes" obtained by treating the dephospho form of phosphoglucomutase with inorganic vanadate in the presence of either glucose 1-phosphate or glucose 6-phosphate, as described in an accompanying paper [Ray, W. J., Jr., Burgner, J. W., II, & Post, C. B. (1990) Biochemistry (second of four papers in this issue)], is centered at a wavelength of 312 nm. The position of this peak amounts to a change in oscillator frequency of about -5000 cm-1 relative to that of tetrahedral VO4(3-). To provide a rationale for this spectral change, the near ultraviolet spectra of the di- and monoanions of inorganic vanadate and a number of derivatives of these anions are compared with that of vanadium (V) in the enzymic complexes, in terms of both what is observed experimentally and what is expected from crystal field theory. Comparisons in water and in largely anhydrous solvents show that water is not an essential element in the coordination sphere of inorganic vanadate or its mono- or diesters and hence that the coordination number of V(V) in such compounds likely is four. These comparisons also show that loss of solvating water from a 4-coordinate vanadate on binding cannot provide a rationale for the spectra of the enzymic complexes. Other comparisons show that neither the binding of metal ions nor protonation nor the binding of vanadate at a site with an unusually high or an unusually low dielectric constant can provide such a rationale. Further comparisons with vanadates known to be pentacoordinate strongly suggest that the coordination number of V(V) in the transition-state-analogue complexes of phosphoglucomutase does not exceed four. In fact, from the standpoint of crystal field theory the marked red shift observed in the electron-transfer absorbance spectrum of the oxyvanadium constellation in these complexes is more reasonably interpreted in terms of a decreased coordination at vanadium (V), viz., in terms of a weakened bonding between vanadium and one or more of its coordinating oxygens. This decreased coordination could be produced by a physical stretching of the vanadate ester linkage. By contrast, the near ultraviolet spectrum of the transition-state-analogue complex that ribonuclease forms with an adduct of uridine and vanadate [Lindquist, R. N., Lynn, J. L., & Lienhard, G. E. (1973) J. Am. Chem. Soc. 95, 8762] is similar to spectra of pentacoordinate model compounds of vanadium(V).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The oxyvanadium constellation in transition-state-analogue complexes of phosphoglucomutase and ribonuclease. Structural deductions from electron-transfer spectra. 214 Jun 98

The three-dimensional structures of ribonuclease (RNase) T1 complexes with the inhibitors 2'-guanylic acid (2'-GMP), 3'-guanylic acid (3'-GMP), and 5'-guanylic acid (5'-GMP) were predicted by energy minimization studies. It is shown that these inhibitors can bind to RNase T1 in either of the ribose puckered conformations (C2'-endo and C3'-endo) in solid state and exist in significant amounts in both forms in solution. These studies are in agreement with the x-ray crystallographic studies of the 2'-GMP-Lys25-RNase T1 complex, where the inhibitor binds in C2'-endo puckered conformation. These results are also in good agreement with the available 1H-nmr results of Inagaki et al. [(1985) Biochemistry 24, 1013-1020], but differ from their conclusions where the authors favor only the C3'-endo ribose conformation for all the three inhibitors. The calculations explain the apparent discrepancies in the conclusions drawn by x-ray crystallographic and spectroscopic studies. An extensive hydrogen-bonding scheme was predicted in all the three complexes. The hydrogen-bonding scheme predicted for the 2'-GMP (C2'-endo)-RNase T1 complex agrees well with those reported from x-ray crystallographic studies. In all three complexes the base and the phosphate bind in nearly identical sites independent of the position of the phosphate or the ribose pucker. The glycosyl torsion angle favors a value in the +syn range in the 2'-GMP (C2'-endo)-RNase T1, 3'-GMP (C2'-endo)-RNase T1, and 3'-GMP (C3'-endo)-RNase T1 complexes; in the high-syn range in the 2'-GMP (C3'-endo)-RNase T1 complex; and in the -syn range in the 5'-GMP (C2'-endo)-RNase T1 and 5'-GMP (C3'-endo)-RNase T1 complexes. These results are in agreement with experimental studies showing that the inhibitory power decreases in the order 2'-GMP greater than 3'-GMP greater than 5'-GMP, and they also explain the high pKa value observed for Glu58 in the 2'-GMP-RNase T1 complex.
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PMID:Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes. 217 61

We previously demonstrated that pneumococcal extracts contain a highly specific inhibitor of human neutrophil elastase (HNE). We now show that the active inhibitor in these extracts is a high-molecular-weight, heat-stable substance that appears to be RNA, since inhibitory activity of pneumococcal extracts is decreased by incubation with ribonuclease but not by incubation with deoxyribonuclease or proteinase K. Moreover, metabolically labeled ([3H]uridine) pneumococcal RNA, isolated by phenol extraction followed by ethanol precipitation, strongly inhibits HNE. Pneumococcal capsular polysaccharide, although polyanionic, is only weakly inhibitory toward HNE and is not a major source of elastase-inhibitory activity in pneumococcal extracts. On the other hand, the capsule of Haemophilus influenzae type b contains polyribosylribitol phosphate. This highly charged polyanion possesses HNE-inhibitory activity, but only under special circumstances to be discussed below. Pneumococci (type I, type II smooth, type II rough) and H. influenzae (type b) all release HNE-inhibitory activity into their culture medium during growth. By contrast, Klebsiella pneumoniae and Staphylococcus aureus release little (if any) stable HNE-inhibitory activity during growth. We propose that some bacterial pneumonias may spare host tissue because polyanions released by the invading microorganisms (e.g. RNA from autolysing pneumococci) inhibit elastase released from inflammatory neutrophils and thereby modulate accompanying tissue proteolysis. Pneumonias caused by microorganisms that do not release stable polyanionic inhibitors of HNE (e.g., Staphylococcus and Klebsiella) may be correspondingly more injurious to the lung.
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PMID:Inhibition of human neutrophil elastase by bacterial polyanions. 244 47

Evidence for capped poly(A) leaders of variable lengths located immediately upstream of the translation initiation codon was obtained by direct analyses of a major late mRNA species. A decapping-recapping method was used to specifically substitute a radioactively labeled phosphate for an unlabeled one within the cap structure. RNase H-susceptible sites were made by hybridizing synthetic oligodeoxyribonucleotides to the mRNA encoding a late major structural protein of 11 kilodaltons. Sequences of the type m7G(5')pppAmp (Ap)nUpG. . ., where n varies from a few to more than 40 nucleotides, were deduced by analysis of the length and sequence of RNase H, RNase T1, and RNase U2 digestion products.
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PMID:Capped poly(A) leaders of variable lengths at the 5' ends of vaccinia virus late mRNAs. 246 59

The preparation of oligouridylic acids was achieved by the stepwise partial hydrolysis of RNA using enzymatical and chemical methods of degradation followed by chromatographic purification steps. After RNA is submitted first to RNase T1 and after that to RNase U2 it is cleaved into oligopyrimidine nucleotides carrying purine nucleotides only at their 3'-terminal. By chemical deamination the pyrimidine sequences are converted to oligouridine sequences. After enzymatical dephosphorylation of the oligouridylic acids their 3'-terminal purine nucleosides are eliminated by a periodate-lysine treatment. The phosphate groups remaining at the 3'-terminal of the resulting oligouridylic acids are enzymatically removed. As a result of the partial hydrolysis the mean recoveries of the particular homologues are U2-U7 93% and U8 86% adding up to about 0.5% of the amount of RNA used as starting material.
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PMID:[Preparation of homologs of oligoribouridylic acid by selective partial hydrolysis of RNA]. 246 92


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