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

Abstract

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.

Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

“…For some members of the REase family, a two-metalion mechanism has been proposed on the basis of analyses of X-ray crystal structures (Lambert et al, 2008;Deibert et al, 2000). Recently, an improved mechanism was proposed in which the ion at site A is implicated to be sufficient for catalysis, while the presence of the second ion greatly accelerates the reaction (Xie et al, 2008). The results of our studies are consistent with this mechanism.…”

“…The number of metal ions retained at the active site in these structures varies; therefore, mechanisms with one metal ion, two metal ions and even three metal ions have independently been proposed (Pingoud et al, 2005). While kinetic analysis with computer simulations suggests that one metal ion is sufficient for the reaction to progress (Pingoud et al, 2009;Xie et al, 2008), it is still unclear whether the divalent metal ions in these multiple sites observed in the crystal structure are critical for the catalytic reaction of REases (Prasannan et al, 2010;Pingoud et al, 2014).…”

ESR spectroscopy identifies inhibitory Cu 2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

“…Positive cooperativity for Mg 2þ interaction has also been observed for restriction endonuclease PvuII, which binds two metal ions in each of its two catalytic sites (42,43); Hill coefficients of n ¼ 3.6 to 4 were reported (44,45). Although the precise role of the second metal ion is in general controversial for many metallonucleases (17,35,39,40), for PvuII the first Mg 2þ bound in a catalytic site is both necessary and sufficient for catalysis (45). The second Mg 2þ acts as a powerful accelerant for DNA cleavage.…”

“…The observed value of n ¼ 1.8-1.9, given that EcoRI uses one metal ion per catalytic center (11,14,17,41), implies interaction between the single-Mg 2þ binding sites of the protein homodimer. Positive cooperativity for Mg 2þ interaction has also been observed for restriction endonuclease PvuII, which binds two metal ions in each of its two catalytic sites (42,43); Hill coefficients of n ¼ 3.6 to 4 were reported (44,45). Although the precise role of the second metal ion is in general controversial for many metallonucleases (17,35,39,40), for PvuII the first Mg 2þ bound in a catalytic site is both necessary and sufficient for catalysis (45).…”

Redesigning the single‐chain variant of the restriction endonuclease PvuII by circular permutation

“…The restriction endonuclease PvuII, thoroughly investigated in terms of structure [16,17] and function [18,19], is a homodimeric enzyme ($18 kDa per monomer) and recognizes and cleaves the palindromic DNA sequence 5 0 -CAG;CTG-3 0 , introducing a specific double-strand break as indicated. By covalently linking the C-terminus of one monomer to the N-terminus of the other one via a short peptide linker, the homodimeric PvuII wild-type was converted into the single-chain variant of PvuII (scPvuII), with a similar DNA cleavage activity and specificity [20].…”