Inhibition of EcoRV endonuclease by deoxyribo-3 '-S-phosphorothiolates: A high-resolution X-ray crystallographic study

Three high-resolution structures of the restriction endonuclease EcoRV boun d to a duplex DNA substrate analogue with deoxyribo-3'-S-phosphorothiolate linkages at both scissile phosphates are presented. In each of these struct ures cocrystallized with Mg2+, Mn2+, or Ca2+ ions, the nonesterified pro-S oxygen of the scissile phosphate no longer directly ligates a divalent cati on, as is observed for the unmodified complex. Instead, one metal ion in al l three structures is shifted toward the adjacent 3'-phosphate of the DNA, to occupy a position nearly identical to that previously observed in an Ero RV T93A/DNA/Ca2+ complex (N. C. Horton et al.. Proc. Natl. Acad Sci. U.S.A. 1998, 95, 13489). A second divalent metal ion in each structure bridges th e carboxylate groups of Asp74 and Glu45 (74/45 site), as also seen in both wild-type and T93A cocrystals. The uncleaved 3'-S-phosphorothiolate DNAs in these complexes are only slightly distorted from the conformation of the u nmodified duplex. Kinetic measurements show that the rate of the chemical s tep For analogue cleavage is severely reduced for each of the active metals Mg2+, Mn2+, and Co2+, and that the thiophilic Mn2+, Cd2+, and Zn2+ cations do not provide a measurable reconstitution of activity. The inability of t hiophilic metals to improve activity is consistent with models for catalysi s derived from previous crystal structures, which indicate that ligation of a metal ion to the 3'-oxygen is mediated through an inner-sphere water mol ecule rather than by direct interaction. The structures suggest that 3'-S-p hosphorothiolale analogues resist cleavage because the bridging sulfur excl udes inner-sphere ligation of divalent metal ions to any position on the sc issile phosphate. This distinguishes the inhibitory mechanism in EcoRV from that operative in the 3'-5' exonuclease active site of DNA polymerase I (C . A. Brautigam et al., Biochemistry, 1999, 38, 696), and likely as well fro m other enzymes which also catalyze phosphoryl transfer via direct metal li gation to the 3'-oxygen leaving group.