| Abstract: | The interaction of zinc with different forms of DNA (λ phage DNA, ss-oligo, ds-oligo) and Mung Bean Nuclease was studied by voltammetric techniques in order to investigate the mechanism of DNA cleavage catalyzed by a zinc metalloenzyme. Stoichiometry, dissociation constant, zinc binding sites and functions were determined for these systems. Two zinc ions were found to be involved in stabilization of a 19 mer ds-oligodeoxyribonucleotide, which was synthesized by the phosphoramidite method and used as a DNA model in the studies. Three zinc ions (Zn1, Zn2, and Zn3), which have different roles in ds-oligo cleavage, were identified in the active site of Mung Bean Nuclease. A concerted SN2 mechanism, which assigns a catalytic function to Zn2 and structural functions to Zn1 and Zn3, was proposed. The hydrolysis of phosphodiester bonds proceeds with inversion of configuration at the phosphorus center, forming a pentacoordinate transition state, which is stabilized by an arginine. Zn2 supplies the nucleophile, which is oriented by an aspartic acid, and activates the ds-oligo by its coordination to the phosphate free oxygen of the phosphodiester bond. Zn1 and Zn3 ions, besides stabilizing the tertiary structure of Mung Bean Nuclease, bind to the leaving group, blocking the cleavage reverse reaction. |
