Groll D H, Jeltsch A, Selent U, Pingoud A
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany.
Biochemistry. 1997 Sep 23;36(38):11389-401. doi: 10.1021/bi9705826.
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.
目前存在两种关于限制性内切酶EcoRV催化机制的模型，这两种模型在直接参与催化的金属离子数量和功能上有所不同。在一种模型中，由Glu45、Asp74和Asp90结合的两个金属离子被认为具有直接催化功能；在另一种模型中，只有一个由Asp74和Asp90结合的金属离子。我们在此表明，在存在Mn2+的情况下，与野生型EcoRV相比，EcoRV - E45A突变体的催化活性仅略有降低（1.8倍），并且E45A切割DNA的单周转速率常数仅降低39倍，而D74A和D90A突变体在所有条件下均无催化活性。这些发现使得Glu45具有重要催化功能（如结合必需二价金属离子）的可能性不大。此外，我们分析了EcoRV和EcoRV突变体切割DNA的速率对Mg2+和Mn2+浓度的依赖性。我们发现，对于野生型酶，DNA切割速率对Mg2+或Mn2+浓度呈S形依赖性，这表明至少有两个金属离子参与DNA结合和催化。然而，这并不意味着EcoRV在DNA切割中遵循双金属离子机制，因为对于E45A突变体，也发现DNA切割速率对Mg2+浓度呈S形依赖性，这使得金属离子与E45/D74位点结合的可能性不大。相比之下，Y219C突变体表现出双曲线依赖性。与早期获得的结果一致，这些发现证明在受Tyr219影响的位点存在Mg2+离子结合，Tyr219是一个远离活性位点的氨基酸残基。该位点的金属结合没有催化作用，而是支持特异性DNA结合。我们得出结论，根据我们的数据，EcoRV不太可能通过双金属离子机制切割DNA，观察到的复杂金属离子效应是由于金属离子结合在不直接参与催化的位点上。
