METAL ION-MEDIATED SUBSTRATE-ASSISTED CATALYSIS IN TYPE-II RESTRICTION ENDONUCLEASES

The 2.15-Angstrom resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent met als bound in one of the active sites. One of these metals is ligated t hrough an inner-sphere water molecule to the phosphate group located 3 ' to the scissile phosphate. A second inner-sphere mater on this metal is positioned approximately in-line for attack on the scissile phosph ate, This structure corroborates the observation that the pro-S-P phos phoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and Bam HI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construc tion of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site a nd invokes assistance in the bond-making step hp a conserved lysine, w hich stabilizes the attacking hydroxide ion nucleophile.