EcoRV endonuclease

Although they often share little sequence similarity and have quite different specificiities, many restriction enzymes have similar three-dimensional structures as well as mechanisms of action. This is true for the EcoRI, BamHl (Fig. 26-5),83/90 EcoRV,91/91a and C/r 101 enzymes,84 and presumably many others. The specifically shaped and tightly packed active sites in the enzyme-substrate complexes ensure specificity. For example, the EcoRV endonuclease cleaves DNA at its recognition site at least a million times faster than at any other DNA sequence.91 As mentioned in Chapter 12, restriction endonucleases require a metal ion, preferably Mg2+, and probably act via a hydroxyl ion generated from Mg2+-OH2 at the active site. Three conserved active site residues, Asp 91, Glu 111, and Lys 113, in the EcoRI endonuclease interact with the DNA near the cleavage site. Lys 113 is replaced by Glu 113 in the BamHl enzyme.83 90... 

Figure 9.42. Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna. The...

F.K. Winkler, D.W. Banner, C. Oefiier, D. Tsemoglou, R.S. Brown, S.P. Heathman, R.K. Bryan, P.D. Martin, K. Petratos, and K.S. Wilson. 1993. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments EMBO J. 12 1781-1795. (PubMed)... 

D. Kostrewa and F.K. Winkler. 1995. Mg + binding to the active site of EcoRV endonuclease A crystallographic study of complexes with substrate and product DNA at 2 A resolution Biochemistry 34 683-696. (PubMed)... 

M.D. Sam and J.J. Perona. 1999. Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease Biochemistry 38 6576-6586. (PubMed)... 

The recognition sequence for EcoRV endonuclease (left) and the sites of methylation (right) in DNA protected from the catalytic action of the enzyme. 

Figure 9.35 Stereochemistry of cleaved DNA. Cleavage of DNA by EcoRV endonuclease results in overall inversion of the stereochemical configuration at the phosphorus atom, as indicated by the stereochemistry of the phosphorus atom bound to one bridging oxygen atom, one 0. one and one sulfur atom. This configuration strongly suggests that the hydrolysis takes place by waters direct attack at the phosphorus atom.

Figure 9.36 A magnesium ion-binding site in EcoRV endonuclease. The magnesium ion helps to activate a water molecule and positions it so that it can attack the phosphorus atom.

Figure 9.42 Greater binding energy of EcoRV endonuclease bound to cognate versus noncognate DNA. The additional interactions between EcoRV endonuclease and cognate DNA increase the binding energy, which can be used to drive DNA distortions necessary for forming a catalytically competent complex.

Some enzymes discriminate between potential substrates by binding them Wxth different affinities. Others may bind many potential substrates but promote chemical reactions efficiently only on specific molecules. Restriction endonucleases such as EcoRV endonuclease employ... 

Hydrogen bonding interactions between EcoRV endonuclease and its DN.A substrate Figure 9,39... 

J. J. Perona and A. M. Martin, Conformational transitions and structural deformabiHty of EcoRV endonuclease revealed by crystallographic analysis, /. Mol. Biol, 1997, 273, 207-225 (pdb lazO). 

Our QM/MM calculations of the minimum-energy catalytic pathways indicate that the catalytic strategy common to both EcoRV endonuclease and myosin ATPase can be formally seen as three-pronged approach. 

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