THE STEREOCHEMICAL COURSE OF THE RESTRICTION ENDONUCLEASE ECORI-CATALYZED REACTION

Abstract

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analog of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues. Performing the reaction in H218O 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] deoyadenosine 5''0-(1-thiotriphosphateo. 31P NMR spectroscopy shows the 18O in this compound to be in a bridging position between the .alpha.- and .beta.-P atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at P. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at P without involvement of a covalent enzyme intermediate.