Does the Restriction Endonuclease EcoRV Employ a Two-Metal-Ion Mechanism for DNA Cleavage? ² Detlef H. Groll, Albert Jeltsch, Ursel Selent, and Alfred Pingoud* Institut fu ¨ r Biochemie, Fachbereich Biologie, Justus-Liebig-UniVersita ¨ t, Giessen, Germany ReceiVed March 13, 1997; ReVised Manuscript ReceiVed July 3, 1997 X ABSTRACT: 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 Mn 2+ , 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 Mg 2+ and Mn 2+ . We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg 2+ or Mn 2+ , 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 Mg 2+ 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 Mg 2+ 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. The type II restriction endonuclease EcoRV (Kholmina et al., 1980) is a highly specific endonuclease that cleaves double-stranded DNA containing GATVATC sequences as indicated (Schildkraut et al., 1984). Like all restriction endonucleases [see reviews by Roberts and Halford (1993), Aggarwal (1995), and Pingoud and Jeltsch (1997)] this enzyme only cleaves DNA in the presence of Mg 2+ ions or other divalent cations, like Mn 2+ , Ni 2+ , or Co 2+ , but not Ca 2+ . In the presence of Mn 2+ , cleavage also occurs at sites differing in one base pair from the canonical site (“star” sites) (Halford et al., 1986). Interestingly, distinct from many but like a few other type II restriction enzymes [PaeR7 (Ghosh et al., 1990), TaqI (Zebala et al., 1992), Cfr9I (Siksnys & Pleckaityte, 1993)], EcoRV also needs Mg 2+ or other divalent cations, e.g. Ca 2+ , for specific DNA binding (Thielking et al., 1992; Vipond & Halford, 1995). This means that divalent metal ions have functional roles in the enzymatic process as well as in the formation of the specificity- determining contacts between the protein and the DNA. Crystallographic studies including metal ion soaking experi- ments with different divalent cations (Kostrewa & Winkler, 1995) have identified one divalent metal ion binding site formed by Asp74 and Asp90 in the enzyme-DNA complex (Table 1). On the basis of structural and biochemical data as well as a comparison with other restriction endonucleases, there is no reasonable doubt that the Asp74/Asp90 site is essential for DNA cleavage and, hence, that the divalent metal ion bound at this site has a catalytic role (Thielking et al., 1991; Winkler, 1992; Selent et al., 1992; Jeltsch et al., 1992, 1993; Vipond et al., 1995; Grabowski et al., 1995). However, there exist four observations demanding for at least one additional divalent metal ion binding site in the EcoRV- DNA complex: First, in the crystallographic studies with some combinations of divalent metal ions electron density was observed in the catalytic center of EcoRV also at a second site formed by Glu45 and Asp74 (Figure 1a and Table 1). Second, metal ion binding at Asp74/Asp90 is not necessary to mediate Mg 2+ dependent specific DNA binding because the D74A and D90A mutants (Thielking et al., 1992), the D74A/D90A double mutant (Ko ¨hler et al., 1994), as well as the E45A/D74A/D90A triple mutant (Jeltsch et al., 1995) all require Mg 2+ for specific DNA binding. In contrast, Mg 2+ dependent specific DNA binding is severely disturbed by a mutation at amino acid Tyr219, which is more than 15 Å away from the catalytic center of the enzyme, suggesting that an additional metal ion binding site might be located in the vicinity of Tyr219 (Jeltsch et al., 1995). Third, cleavage experiments of phosphorothioate substituted oligonucleotides in the presence of Mg 2+ or Mn 2+ gave evidence for a metal ion binding site at the first phosphate within the recognition sequence (GpATATC), which in the complex is near Tyr219 (Jeltsch et al., 1995). Fourth, the * Address correspondence to this author at Institut fu ¨r Biochemie, Fachbereich Biologie, Justus-Liebig-Universita ¨t, Heinrich-Buff-Ring 58, 35392 Giessen, Germany. Phone: +49 641 99 35400. Fax: +49 641 99 35409. ² This work has been supported by the Deutsche Forschungsgemein- schaft (Pi 122/12-1) and the Fonds der Chemischen Industrie. X Abstract published in AdVance ACS Abstracts, September 1, 1997. 11389 Biochemistry 1997, 36, 11389-11401 S0006-2960(97)00582-5 CCC: $14.00 © 1997 American Chemical Society