Kinetic mode of Eco RI restriction endonuclease catalyzed reaction

Abstract

Type II restriction endonucleases provide a system for the study on the interaction between proteins and specific nucleotide sequences on DNA molecules. We investigated a characteristic mode of the interaction between EcoRI endonuclease and substrates. In the study, the degree of the enzymic reaction was monitored by film scanning technique after separation of the resulting products by agarose. Both superhelical and linear pBR322 DNA showed maximal activity at pH7.5. Therefore, shape difference of substrate did not affect the optimal activity of enzyme as a function of pH. The rate of DNA cleavage by the enzyme was maximal at 37 C and the activation energy ($E_a$) was 8.5 Kcal/mole at pH 7.5 irrespective of superhelical or linear form. The optimal concentration of $Mg^{++}$ for the enzyme activity was found to be 5 mM. Although a little activity difference was exhibited, both substrates showed the same optimal activity at 5 mM $MgCl_2$ concentration. Also both substrates showed same maximal activity at 50 mM NaCl concentration. It was investigated whether or not the shapes of substrates and non-specific binding sites near the recognition site were related to the enzyme-substrate interaction. $K_m$ value of superhelical and linear pBR322 DNA and λ DNA was 5.8 nM, 8.1 nM, and 3.2 nM, respectively. It was shown that superhelical form had the more affinity than linear form to enzyme, and that λ DNA had the more affinity than linear pBR322 DNA to enzyme. The pK values of the prototropic groups at the active site of EcoRI endonuclease were determined by measuring the pH-dependence of $slope_{1/s}$ and $\mbox{V_{max_{app}}}$ using Dixon-Webb plots. The plot was shown for $V_{max}=1 nM$, $PK_{es1}=5.5$, and $pK_{es2}=9.0$. $pK_{es1}$ value was contained to imidazolium (of histidine) pK value range. $pK_{es2}$ value was similar to sulfhydryl (of cysteine) and hydroxyl (of serine) pK value. Based on a series of kinetic experiment, a model mechanism of phosphodiesterase a...