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 bound to a duplex DNA substrate analogue with deoxyribo-3'-S-phosphorothiolate linkages at both scissile phosphates are presented. In each of these structures cocrystallized with Mg2+, Mn2+, or Ca2+ ions, the nonesterified pro-S oxygen of the scissile phosphate no longer directly ligates a divalent cation, as is observed for the unmodified complex. Instead, one metal ion in all three structures is shifted toward the adjacent 3'-phosphate of the DNA, to occupy a position nearly identical to that previously observed in an EroRV 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 the 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 unmodified duplex. Kinetic measurements show that the rate of the chemical step 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 thiophilic metals to improve activity is consistent with models for catalysis derived from previous crystal structures, which indicate that ligation of a metal ion to the 3'-oxygen is mediated through an inner-sphere water molecule rather than by direct interaction. The structures suggest that 3'-S-phosphorothiolale analogues resist cleavage because the bridging sulfur excludes inner-sphere ligation of divalent metal ions to any position on the scissile 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 from other enzymes which also catalyze phosphoryl transfer via direct metal ligation to the 3'-oxygen leaving group.