1H56

Structural and biochemical characterization of a new magnesium ion binding site near Tyr94 in the restriction endonuclease PvuII

Summary for 1H56

• Entry DOI: 10.2210/pdb1h56/pdb
• Related: 1EYU 1F0O 1K0Z 1NI0 1PVI 1PVU 2PVI 3PVI
• Descriptor: TYPE II RESTRICTION ENZYME PVUII, MAGNESIUM ION (3 entities in total)
• Functional Keywords: endonuclease, type ii restriction endonuclease, hydrolase, nuclease
• Biological source: PROTEUS VULGARIS
• Total number of polymer chains: 2
• Total formula weight: 36528.21

Authors

Spyrida, A.,Matzen, C.,Lanio, T.,Jeltsch, A.,Simoncsits, A.,Athanasiadis, A.,Scheuring-Vanamee, E.,Kokkinidis, M.,Pingoud, A. (deposition date: 2001-05-20, release date: 2003-08-07, Last modification date: 2023-12-13)

Primary citation

Spyridaki, A.,Matzen, C.,Lanio, T.,Jeltsch, A.,Simoncsits, A.,Athanasiadis, A.,Scheuring-Vanamee, E.,Kokkinidis, M.,Pingoud, A.
Structural and Biochemical Characterization of a New Mg(2+) Binding Site Near Tyr94 in the Restriction Endonuclease PvuII.
J.Mol.Biol., 331:395-, 2003

PubMed Abstract: 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.

PubMed: 12888347
DOI: 10.1016/S0022-2836(03)00692-2

Experimental method

X-RAY DIFFRACTION (3 Å)