title: Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.
abstract: The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined. This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases. The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease. Performing this reaction in H2 18O gave [18O]dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to [18O]dAMPS with nuclease P1. This deoxynucleoside 5'-[18O]phosphorothioate was stereospecifically converted to [18O]dATP alpha S with adenylate kinase and pyruvate kinase [Brody, R. S., & Frey, P. A. (1981) Biochemistry 20, 1245-1251]. Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms. This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus. The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state. A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate. An identical result has been previously observed with the EcoR1 endonuclease [Connolly, B. A., Eckstein, F., & Pingoud, A. (1984) J. Biol. Chem. 259, 10760-10763]. X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites. Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.
authors.affiliation: Department of Biochemistry, University of Southampton, U.K.
authors.forename: J A
authors.initials: JA
authors.lastname: Grasby
authors.forename: B A
authors.initials: BA
authors.lastname: Connolly
mesh_terms: D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010758:Phosphorus / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D009838:Oligodeoxyribonucleotides; D010758:Phosphorus; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium