Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn²⁺, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg²⁺ and Mn²⁺. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg²⁺ or Mn²⁺, indicative of at least two metal ions involved in DNA binding and catalysis. This, however, does not mean that EcoRV follows a two-metal-ion mechanism in DNA cleavage, because also for the E45A mutant a sigmoidal dependence of the rate of DNA cleavage on the Mg²⁺ concentration was found, making metal ion binding to the E45/D74 site unlikely. In contrast, the Y219C mutant shows a hyperbolic dependence. In agreement with results obtained earlier, these findings demonstrate binding of a Mg²⁺ ion at a site influenced by Tyr219, an amino acid residue that is far away from the active site. Metal binding at this site does not have a catalytic role but rather supports specific DNA binding. We conclude that on the basis of our data a two-metal-ion mechanism of DNA cleavage is unlikely for EcoRV and that the complex metal ion effects observed are due to metal ion binding at sites that are not directly involved in catalysis.

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