http://purl.uniprot.org/citations/12888347 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
http://purl.uniprot.org/citations/12888347 | http://www.w3.org/2000/01/rdf-schema#comment | "We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.org/dc/terms/identifier | "doi:10.1016/s0022-2836(03)00692-2"xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Jeltsch A."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Pingoud A."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Athanasiadis A."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Kokkinidis M."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Simoncsits A."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Lanio T."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Matzen C."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Scheuring-Vanamee E."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/author | "Spyridaki A."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/date | "2003"xsd:gYear |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/name | "J Mol Biol"xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/pages | "395-406"xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/title | "Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII."xsd:string |
http://purl.uniprot.org/citations/12888347 | http://purl.uniprot.org/core/volume | "331"xsd:string |
http://purl.uniprot.org/citations/12888347 | http://www.w3.org/2004/02/skos/core#exactMatch | http://purl.uniprot.org/pubmed/12888347 |
http://purl.uniprot.org/citations/12888347 | http://xmlns.com/foaf/0.1/primaryTopicOf | https://pubmed.ncbi.nlm.nih.gov/12888347 |
http://purl.uniprot.org/uniprot/#_P23657-mappedCitation-12888347 | http://www.w3.org/1999/02/22-rdf-syntax-ns#object | http://purl.uniprot.org/citations/12888347 |
http://purl.uniprot.org/uniprot/P23657 | http://purl.uniprot.org/core/mappedCitation | http://purl.uniprot.org/citations/12888347 |