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

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pancreatic ribonuclease A (RNase A) is a endonuclease that catalyzes depolymerization of ribonucleic acid (RNA) releasing oligonucleotides. In the process of binding enzyme with substrate are involved several non-catalytic phosphate binding subsites, one of them is p2, additional to main catalytic site p1. RNaza A prefers binding and cleavage of longer substrate molecules, and 3',5'-phosphodiester bond should be some six-seven residues apart from the end of molecules of the chain of RNA. In this work is analysed endonuclease activity of recombinant pancreatic RNase A (K7H), that in position seven instead of a lysine there is a histidine, amino acid residue that participates in main catalytic site p1. Mutant enzyme is obtained by site-directed mutagenesis by Kunkel. Results of this investigation have shown that substitution of lysine by histidine in position seven of RNase A has produced total deletion of p2 subsite, and K7H has lost endonuclease activity, and has become exonuclease. These results confirm central role of Lys-7 in establishing p2 subsite and endonuclease activity of pancreatic RNase A.
...
PMID:[Endonuclease activity of recombinant pancreatic nuclease (A-K7H)]. 1038 39

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.
...
PMID:Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs. 1043 89

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.
...
PMID:NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9. 1045 17

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.
...
PMID:Charge-to-alanine mutagenesis of the adeno-associated virus type 2 Rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants. 1051 52

A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.
...
PMID:A novel endonuclease mechanism directly visualized for I-PpoI. 1058 47

Homing endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with extremely high specificity. I-PpoI endonuclease, a homing endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) of known three-dimensional structure that catalyzes the cleavage of a long target DNA sequence (15 base pairs). Here, a detailed chemical mechanism for catalysis of DNA cleavage by I-PpoI endonuclease is proposed and tested by creating six variants in which active-site residues are replaced with alanine. The side chains of three residues (Arg61, His98, and Asn119) are found to be important for efficient catalysis of DNA cleavage. This finding is consistent with the proposed mechanism in which His98 abstracts a proton from an attacking water molecule bound by an adjacent phosphoryl oxygen, Arg61 and Asn119 stabilize the pentavalent transition state, and Asn119 also binds to the essential divalent metal cation (e.g., Mg(2+) ion), which interacts with the 3'-oxygen leaving group. Because Mg(2+) is required for cleavage of a substrate with a good leaving group (p-nitrophenolate), Mg(2+) likely stabilizes the pentavalent transition state. The pH-dependence of k(cat) for catalysis by I-PpoI reveals a macroscopic pK(a) of 8.4 for titratable groups that modulate product release. I-PpoI appears to be unique among known restriction endonucleases and homing endonucleases in its use of a histidine residue to activate the attacking water molecule for in-line displacement of the 3'-leaving group.
...
PMID:Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI. 1058 40

Homing endonucleases are classified into four families based on active site sequence motifs. Through structural comparisons we have found structural similarities between the endonuclease domain of colicin E9, an H-N-H motif-containing enzyme, and both the non-specific nuclease from Serratia and I-PpoI, a His-Cys box-containing homing endonuclease. Our comparison identifies conservation at the heart of all three enzyme active sites and so argues for a re-classification of H-N-H and His-Cys box homing endonucleases as a single family. We suggest the 'betabetaalpha-Me family' of homing enzymes to reflect the three elements of secondary structure and the metal ion that define the motif.
...
PMID:Structural parsimony in endonuclease active sites: should the number of homing endonuclease families be redefined? 1060 25

The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3'-exonuclease activity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease activities. Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His(371) with Ala. The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability. In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His(371) is the active-site residue that has an essential role in PRV DNase activity.
...
PMID:Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase. 1067 64

The nuclear tRNA 3' processing activity from wheat has been characterized and partially purified. Several characteristics of the wheat nuclear 3' processing enzyme now allow this activity to be distinguished from its mitochondrial counterpart. The nuclear enzyme is an endonuclease, which we termed nuclear RNase Z. The enzyme cleaves at the discriminator base and seems to consist only of protein subunits, since essential RNA subunits could not be detected. RNase Z leaves 5' terminal phosphoryl and 3' terminal hydroxyl groups at the processing products. It is a stable enzyme being active over broad temperature and pH ranges, with the highest activity at 35 degrees C and pH 8.4. The apparent molecular mass according to gel filtration chromatography is 122 kDa. The nuclear RNase Z does process 5' extended pretRNAs but with a much lower efficiency than 5' matured pretRNAs. Nuclear intron-containing precursor tRNAs as well as mitochondrial precursor tRNAs are efficiently cleaved by the nuclear RNase Z. Mitochondrial pretRNA(His) is processed by the nuclear RNase Z, generating a mature tRNA(His) containing an 8 base pair acceptor stem. The edited mitochondrial pretRNA(Phe) is cleaved easily, while the unedited version having a mismatch in the acceptor stem is not cleaved. Thus, an intact acceptor stem seems to be required for processing. Experiments with precursors containing mutated tRNAs showed that a completely intact anticodon arm is not necessary for processing by RNase Z. Comparison of the plant nuclear tRNA 3' processing enzyme with the plant mitochondrial one suggests that both activities are different enzymes.
...
PMID:tRNA 3' processing in plants: nuclear and mitochondrial activities differ. 1068 60

Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity.
...
PMID:Endonuclease activity in lipocalins. 1076 87


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---