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| | {{STRUCTURE_1h56| PDB=1h56 | SCENE= }} | | {{STRUCTURE_1h56| PDB=1h56 | SCENE= }} |
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| - | '''STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF A NEW MAGNESIUM ION BINDING SITE NEAR TYR94 IN THE RESTRICTION ENDONUCLEASE PVUII'''
| + | ===STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF A NEW MAGNESIUM ION BINDING SITE NEAR TYR94 IN THE RESTRICTION ENDONUCLEASE PVUII=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_12888347}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 12888347 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_12888347}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Nuclease]] | | [[Category: Nuclease]] |
| | [[Category: Type ii restriction endonuclease]] | | [[Category: Type ii restriction endonuclease]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 18:26:39 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 06:38:33 2008'' |
Revision as of 03:38, 1 July 2008
Template:STRUCTURE 1h56
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF A NEW MAGNESIUM ION BINDING SITE NEAR TYR94 IN THE RESTRICTION ENDONUCLEASE PVUII
Template:ABSTRACT PUBMED 12888347
About this Structure
1H56 is a Single protein structure of sequence from Proteus vulgaris. Full crystallographic information is available from OCA.
Reference
Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII., Spyridaki A, Matzen C, Lanio T, Jeltsch A, Simoncsits A, Athanasiadis A, Scheuring-Vanamee E, Kokkinidis M, Pingoud A, J Mol Biol. 2003 Aug 8;331(2):395-406. PMID:12888347
Page seeded by OCA on Tue Jul 1 06:38:33 2008
