1h56: Difference between revisions

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.

1H56 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Proteus_vulgaris Proteus vulgaris] with MG as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Type_II_site-specific_deoxyribonuclease Type II site-specific deoxyribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.21.4 3.1.21.4] Structure known Active Site: MGA. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1H56 OCA].  

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