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Three different mechanisms have been proposed to describe DNA cleavage by the type II restriction endonuclease EcoRV, which differ in the number and function of metal ions directly involved in catalysis and the different roles assigned to amino acid residues in the active sites and a phosphate group of the substrate. There are only four acidic amino acid residues close to the scissile bond: the essential Asp74 and Asp90, the non-essential Glu45, and Asp36. We show here that Asp36 can be exchanged for alanine, with only minor effects on the cleavage rate of the nearby phosphodiester bond, excluding that Asp36 could be directly involved in catalysis. Hence, the two versions of the two-metal-ion mechanism are not compatible with the experimental data, because too few ligands for two metal ions are present near the active site of EcoRV. Our result, thus, supports the one-metal-ion mechanism for EcoRV. We suggest that Asp36 has an allosteric effect by which specific contacts between one strand of the DNA and one subunit of the enzyme trigger the activation of one catalytic center. Given the similar structures of the active sites of EcoRV, EcoRI, BamHI, PvuII and FokI, as well as the occurrence of a characteristic catalytic motif in several other restriction enzymes, we conclude that these enzymes most likely share a similar mechanism of DNA cleavage, whose characteristic feature is the involvement of only one Mg2+ ion in catalysis.

biological chemistry

The Mechanism of DNA Cleavage by the Type II. Restriction Enzyme EcoRV: Asp36 Is Not Directly Involved in DNA Cleavage but Serves to Couple Indirect. Readout to Catalysis