title: Using single-turnover kinetics with osmotic stress to characterize the EcoRV cleavage reaction.
abstract: Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg(2+) ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations has not been established. In this work, we employ a single-turnover technique that uses osmotic stress to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg(2+) and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient time scale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg(2+) concentration and osmotic pressure gives the number of Mg(2+) ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5-2.5 Mg(2+) ions, the uptake of ∼30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate-limiting conformational change of each monomer of the dimeric enzyme that allows Mg(2+) ion binding. This modified single-turnover protocol has general applicability for metalloenzymes.
authors: The Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors: Rocco
authors: R
authors: Ferrandino
authors: Nina
authors: N
authors: Sidorova
authors: Donald
authors: D
authors: Rau
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D009997:Osmotic Pressure; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium