Catalytic Roles of Divalent Metal Ions in Phosphoryl Transfer by EcoRV Endonuclease

Abstract

The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

“…Apart from BamHI and BglII, the Mg 2+ concentration optimum occurs at an ∼ 10-fold higher concentration (on average between 1 and 10 mM) than the Mn 2+ concentration optimum (on average between 0.1 and 1 mM) ( Table 2), as has been observed before for some restriction enzymes (e.g., EcoRV). 36 In all cases except BamHI, the maximal DNA cleavage rate in the presence of Mn 2+ is lower than the Mg 2+ -dependent DNA cleavage at optimum conditions. In the majority of cases, the enzymes tested are inhibited by Mg 2+ concentrations of above ∼ 10 mM and by Mn 2+ concentrations exceeding ∼ 1 mM.…”

Mechanistic Insights from the Structures of HincII Bound to Cognate DNA Cleaved from Addition of Mg2+ and Mn2+

“…The crystallization and soaking conditions are at pH 5.5, and while the pH optimum of HincII is not known, that of related enzymes is typically greater than 7. 22 Crystals soaked in either 10 mM MgCl 2 or MnCl 2 did not show any signs of DNA cleavage. The higher concentration (50 mM) of MgCl 2 or MnCl 2 required for DNA cleavage in the crystals may have resulted in better, or faster, penetration of the crystal.…”

“…1,[15][16][17][18][19][20] By far, the greatest number of mechanistic studies of divalent cation-dependent cleavage of duplex DNA by an enzyme have been done with the type II restriction endonucleases. [15][16][17][18][19][20][21][22][23][24]63 Typically, the type II restriction enzymes utilize Mg 2C as the in vivo divalent cation cofactor, but will cleave DNA, albeit with less sequence specificity, in the presence of Mn 2C or Co 2C . 25 In addition, while EcoRI binds specific DNA firmly in the absence, as well as in the presence of divalent metal ions, 26,27 extensive binding studies using the gel-shift assay, filter binding, and fluorescence polarization anisotropy have determined that substitution of Ca 2C for Mg 2C confers specific and tight binding in some restriction endonucleases, such as EcoRV and MunI, while preventing cleavage of the DNA.…”

DNA Cleavage by EcoRV Endonuclease: Two Metal Ions in Three Metal Ion Binding Sites

“…This model is supported by pH-rate studies, which show that the Mg 2+ -dependent phosphoryl transfer step follows a sharp bellshaped curve with an optimum at pH 8.5, suggesting general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3′-O anion by a second ionized water (29). However, a weakness of the model is that all three divalent metal sites have not been simultaneously observed in any single structure (25,26).…”

“…This suggests that the active site environment favors a positive charge at this position, helping to elucidate the role of this conserved lysine in catalysis. FIGURE 1: Three-metal mechanism for the transition state of DNA cleavage by EcoRV (24,29). The metal ion located in site I generates the attacking hydroxide nucleophile, which is stabilized and oriented by the positively charged Lys92.…”