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You've created a Promega. Check your inbox to complete email verification. Note: You will not be able to access your account until your email is verified. Our records indicate that this email address is already registered. There was an issue creating your account. There was an error processing your request. The information is filed in different subsections. Length: Mass Da : 34, It is useful for tracking sequence updates.
The algorithm is described in the ISO standard. Full view. Streptococcus downei MFe These are stable identifiers and should be used to cite UniProtKB entries. Upon integration into UniProtKB, each entry is assigned a unique accession number, which is called 'Primary citable accession number'.
See complete history. Do not show this banner again. Endonuclease , Hydrolase , Nuclease. BioCyc i. Restriction enzymes and methylases database More Search reactions for this EC number in Rhea. Short name: R. This is known as the 'taxonomic identifier' or 'taxid'. It lists the nodes as they appear top-down in the taxonomic tree, with the more general grouping listed first. ChEMBL i. DrugCentral More DrugCentral i.
BindingDB database of measured binding affinities More The presence of the His-tag sequence caused no gel shift: no DNA bound to enzyme data not shown. It is possible that the positive charges of the N-terminal His residues might bind DNA in an arbitrary manner and hinder the correct interaction. Unmodified enzymes cause a gel shift, as illustrated in Figure 4A. Mutants VE and KN showed weak affinity in comparison with wild type. In Table II , we summarize relative intensities in other words, affinity to the probe of the shifted bands.
A small amount of enzyme was present in the reaction, and it was terminated after 30 min: a shifted band and the reaction product could be observed at once. Radioactivity measurement implied that the unmodified enzyme showed a threefold higher affinity to the probe or substrate than the His-tagged enzyme. As described above, mutant DL lost all activity. There is a possibility that the three-dimensional structure of the protein was destroyed in this mutant. Also, the extent of the structural difference between the E86K mutant and that of wild type should be investigated.
The curve gradually decreased from down to nm, showed a negative trough at nm, then rapidly increased in regions below nm. We therefore conclude that the Hin dIII protein did not change its conformation by site-directed mutagenesis, even if the mutant enzymes showed drastic changes in biochemical properties.
It is well known that restriction enzymes require divalent cations as cofactors. We considered the possibility that the mutant E86K allowed water molecules to enter its catalytic site more easily.
This would explain the high activity of this mutant. We examined the effects of several kinds of divalent cations on activities of the wild type enzyme and mutant E86K, as displayed in Figure 6. In Figure 6A , the effects of five kinds of divalent cations were examined. We repeated their experiment using commercial or our purified wild type enzymes, and obtained a loss of activity, again.
This additional activity of E86K was exhibited with the linear plasmid, pHAA Eco RI, more efficiently than with the intact circular plasmid data not shown.
As shown in Figure 6A and B , the normal product band of 1. The bands corresponding to 1. Respective five of the emerged white colonies yielded plasmids, which were all found to have inserts of 1. The results in Figure 6B are similar to those in Figure 6A. The band marked with an asterisk 0.
This activity was attained only in E86K, with a coexistent transition metal. No such bands were found when pUC19 was used. The bands marked with an asterisk in Figures 6B and 7 were both recovered and purified. Both of them had UV absorption peaks at nm. All the substrate DNAs were prepared by CsCl density gradient ultracentrifugation: no contaminating bacterial nucleic acids were present.
Although their sequences have not yet been determined, we think this activity by E86K would not be regarded as a so-called star activity. These facts show that the altered specificity of E86K is different from star activity. In general, the smaller the ion radius, the higher the hydration number.
Because of its small radius about 0. We therefore think that the novel action of E86K is not related to ion radius or hydration of cations: it is specifically caused by transition metals, which cannot be replaced with the usual divalent cations.
A mutational analysis of the Klenow fragment of E. For example, a mutant of restriction enzyme Eco RI, in which Glu was replaced with Gly, showed a reduced activity King et al. One can strongly argue that Asp is directly responsible for the activity: it is one of the two acidic residues at the catalytic center, like Glu in Eco RI.
Our model as to the role of transition metals is as follows. Substitution of Lys for Glu at residue 86 would bring about a conformation which permits the second cation, especially that of a transition metal ion, to bind another phosphate group s to be attacked by water.
This is the first report describing a change of substrate specificity for a restriction enzyme under moderate conditions. It is hoped that more optimal conditions for the E86K treatment will be discovered soon to shed light on the complete alteration of substrate specificity of Hin dIII.
Two mutants, VE and KN, were prepared to examine the effect of mutations introduced in the vicinity of Asp and Asp Introduction of negative charge VE and the disappearance of positive charge KN resulted in a decrease in both enzyme and DNA binding activities. However, these mutants are not as remarkably inactive as DL. It may be the case that the Val and Lys residues are not members of the motif proposed by Stahl et al. Investigation of the three-dimensional structure of Hind III is necessary for further discussion.
Table I. Oligonucleotides used for PCR cloning and mutagenesis. New restriction sites are underlined, and replaced codons are indicated by lowercase letters. M, molecular weight markers; sizes are illustrated on left side.
Hin dIII activities of purified enzymes. A Effect of the His-tag at the N-terminus of the wild type enzyme. Enzymes: lane 1, commercial enzyme; 2 and 3, His-tagged 0. B Activiites of mutant enzymes. C Comparison of activities of unmodified dilute enzymes.
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