One and Two Metal Ion Catalysis: Global Single Turnover Kinetic Analysis of PvuII Endonuclease Mechanism

摘要

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of metallonuclease mechanism. DNA association rate constants are about 100-fold higher in the presence of the catalytically nonsupportive Ca(II) vs. the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM2S and EM4S) support cleavage. Interestingly, the cleavage rate for EM2S is about 100-fold slower than that displayed by EM4S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.