The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction.

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.