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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli [Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P. and Vanoni, M. (1995) Gene 154, 99-103]. NMR and photo-CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster consisting of Phe5, Tyr7, Phe31 and Tyr33 while Trp23 is fully exposed to solvent. Phe31, Tyr33 and Trp23 are located within a triple stranded antiparallel beta-sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding. Phe31 and Trp23 are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA-binding proteins.
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PMID:1H-NMR and photo-CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus. 755 54

Proton sharing between acidic groups has been observed in the active sites of several enzymes, including bacteriorhodopsin, aspartic proteases, and ribonuclease HI. We here report NMR observations suggestive of proton sharing between cysteine thiols in the active site of the oxidation-reduction enzyme thioredoxin. The pKas of the two cysteine thiols in the Escherichia coli protein are removed from the expected value of 8.4 by approximately 1 pH unit in either direction, upward and downward. Further, the C beta resonances of both residues show clearly the effects of both of these pKas, indicating that the titrations of the two thiol groups are intimately linked. This behavior strongly suggests that the low pKa ascribed to the deprotonation of the Cys 32 thiol most likely arises through the interaction and close approach of the thiol of Cys 35, with the thiolate anion of Cys 32 stabilized through the sharing of the remaining thiol proton, nominally attached to Cys 35. These observations provide a rationale for the mediation of active site pH control, an important aspect of the mechanism of thioredoxin and other proteins with catalytic thioredoxin domains, such as protein disulfide isomerases.
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PMID:Proton sharing between cysteine thiols in Escherichia coli thioredoxin: implications for the mechanism of protein disulfide reduction. 764 Feb 64

Peptide-water interactions of a ribonuclease C-peptide analogue, RN-24 (Suc-AETAAAKFLRAHANH2), which exhibits significant helicity, have been studied in solution using homonuclear 2D and 3D NMR cross-relaxation experiments. Dipolar peptide proton-water proton interactions are indicated by a large number of NOESY-type cross peaks at the H2O resonance frequency, most of them with opposite sign relative to the diagonal. Some cross peaks arise from intrapeptide cross relaxation to labile protons of histidine, threonine, lysine and arginine side chains. The observed peptide-water interactions are rather uniformly distributed, involving peptide backbone and side chains equally. The data are consistent with rapid fluctuations of the conformational ensemble and the absence of peptide regions that are highly shielded from bulk solvent, even in a peptide that exhibits high propensities for formation of helical secondary structure.
J Biomol NMR 1995 Jun
PMID:Hydration of the partially folded peptide RN-24 studied by multidimensional NMR. 764 54

A useful approach using an MLEV-17 pulse sequence was developed to identify histidine C epsilon 1H magnetic resonances of proteins. This technique can be readily applied to proteins dissolved directly in deuterium oxide solution and eliminates the necessity for an exhaustive exchange of NH to ND. Because of its sensitivity, this technique makes it possible to significantly extend the limitations on protein size. The utility of this spin-lock sequence is demonstrated using ribonuclease, subtilisin, and human prostatic acid phosphatase, with molecular weights ranging from 12K to 100K. With this technique, all three or four of the histidine 1H NMR signals of two human low-molecular-weight phosphotyrosyl protein phosphatases (HCPTP-A or -B, respectively) were readily detected. Histidine peak assignments were accomplished through the use of histidine to alanine mutants of HCPTP-A and -B and a homologous bovine enzyme. Analysis of the pH titration curves of these signals provided microscopic pKa's for the histidines in the human enzymes. A comparison of corresponding histidine pKa values of the two isoenzymes, together with an examination of the 1H NMR spectra of the proteins, provided evidence of significant differences in secondary structure. Titration of HCPTP-A and -B with vanadate, a strongly bound competitive inhibitor, caused the His-72 peak to appear as two signals at nearly equimolar concentrations of protein and vanadate, while the other histidine peaks were not affected. This is interpreted to mean that His-72 is at the enzyme active site.
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PMID:Identification and pKa determination of the histidine residues of human low-molecular-weight phosphotyrosyl protein phosphatases: a convenient approach using an MLEV-17 spectral editing scheme. 768 32

Monomeric bovine seminal ribonuclease (mBS-RNase), the subunit of dimeric bovine seminal ribonuclease (BS-RNase), is an unusual monomer: for its structural stability, its catalytic activity, which is even higher than that of the parent dimeric enzyme, and for its role as an intermediate in the refolding of dimeric BS-RNase. Here we present the proton NMR assignment and secondary-structure determination of mBS-RNase, with a comparison of its structure to the structure of its parent protein, and to the structure of RNase A, a homologue with more than 80% identity in amino acid sequence. Proton NMR assignment was performed using a computer-assisted procedure, through a partially automated analysis of homonuclear three-dimensional spectra [Oschkinat, H., Holak, T. A. & Cieslar, C. (1991) Biopolymers 31, 699-712]. The secondary structures of mBS-RNase, of the A chain of dimeric BS-RNase, and of RNase A, are found to be similar. Significant differences are found instead, between mBS-RNase and RNase A in the more flexible stretches of the molecule, where a higher number of substitutions is present. Furthermore, a preliminary tertiary-structure model is reported, showing that the overall folding of mBS-RNase is closer to that of RNase A rather than that of (dimeric) BS-RNase.
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PMID:Assignment and secondary-structure determination of monomeric bovine seminal ribonuclease employing computer-assisted evaluation of homonuclear three-dimensional 1H-NMR spectra. 774 72

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.
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PMID:Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models. 775 90

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.
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PMID:Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR. 790 91

2,5-Hexanedione (2,5-HD) is the neurotoxic gamma-diketone metabolite of the industrial solvent n-hexane. Substantial evidence indicates that 2,5-HD reacts with neurofilament protein lysine epsilon-amines to yield 2,5-dimethylpyrrole adducts and that this reaction is critical to the mechanism of toxicity. Alkylpyrroles are susceptible to autoxidative dimerization, a process that has also been suggested as an obligatory step in 2,5-HD neuropathy. In the present study, we characterized pyrrole autoxidation products of a 2,5-HD-treated lysine analogue and of a model, lysine-containing dipeptide and examined mechanistic aspects of pyrrole-mediated protein cross-linking. Incubation of 2,5-HD with N alpha-acetyllysine or the dipeptide N alpha-acetylglycyllysine methyl ester in physiological buffer (pH 7.4) under oxidative conditions resulted in time-dependent formation of the N epsilon-pyrrole derivative and two major pyrrole autoxidation products, as demonstrated by HPLC, on-line thermospray MS, and UV photodiode array detection. An autoxidative pyrrole dimer containing a methylene bridge between C-2 of one pyrrole ring and C-3 of a second ring was characterized by thermospray MS and 1H-NMR spectroscopy. 13C-NMR spectroscopy provided evidence for an identical pyrrole-to-pyrrole bridge in autoxidized, pyrrolylated ribonuclease (RNase). MS analysis also revealed a second major product--a stable, oxygen-containing monomeric pyrrole derivative. This product exhibited a UV absorbance maximum (lambda max = 355 nm) consistent with extended conjugation. Polymerization of pyrrolylated acetyllysine was accelerated by persulfate, a free-radical initiator, and inhibited by ascorbate, an antioxidant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Formation and structure of cross-linking and monomeric pyrrole autoxidation products in 2,5-hexanedione-treated amino acids, peptides, and protein. 798 20

Ribonucleases catalyze the hydrolysis of the P-O5' bond in RNA. This reaction occurs in two steps: transphosphorylation of RNA to a 2',3'-cyclic phosphodiester intermediate and hydrolysis of this intermediate to a 3'-phosphomonoester. 31P NMR spectroscopy was used to monitor the accumulation of the 2',3'-cyclic phosphodiester intermediate during the transphosphorylation and hydrolysis reactions catalyzed by various ribonucleases and by small molecules. The intermediate was found to accumulate during catalysis by monomeric bovine pancreatic ribonuclease A (RNase A), a dimer and a trimer of RNase A, bovine seminal ribonuclease, RNase T1, barnase, and RNase 1. These enzymes, which are of widely disparate phylogenetic origin, released rather than hydrolyzed most of the intermediate formed transphosphorylation of RNA. In contrast, the intermediate did not accumulate during catalysis by hydroxide ion or imidazole buffer. In the presence of these small molecules, hydrolysis is faster than transphosphorylation. A trapping experiment was used to assess the throughput of the reaction catalyzed by RNase A. [5,6-3H]Uridylyl-(3'-->5')adenosine was incubated with RNase A in the presence of excess unlabeled uridine 2',3'-cyclic phosphodiester, which dilutes the specific radioactivity of any released cyclic intermediate. Only 0.1% of the RNA substrate was found to be both transphosphorylated and hydrolyzed without dissociating from the enzyme. These results suggest that ribonucleases have evolved primarily to catalyze RNA transphosphorylation and not RNA hydrolysis.
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PMID:Energetics of catalysis by ribonucleases: fate of the 2',3'-cyclic phosphodiester intermediate. 800 6

Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS-RNase and [H119D]BS-RNase) prepared by site-directed mutagenesis. This activity came from a dimer (M x I) with a composite active site. 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly. Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).
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PMID:A misfolded but active dimer of bovine seminal ribonuclease. 807 30


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