| Abstract
| - Mutational analysis has previously indicated that D83 and E98 residues are essential for DNAcleavage activity and presumably chelate a Mg2+ ion at the active site of MunI restriction enzyme. In theabsence of metal ions, protonation of an ionizable residue with a pKa> 7.0, most likely one of the activesite carboxylates, controls the DNA binding specificity of MunI [Lagunavicius, A., Grazulis, S., Balciunaite,E., Vainius, D., and Siksnys, V. (1997) Biochemistry36, 11093−11099.]. Thus, competition between H+and Mg2+ binding at the active site of MunI presumably plays an important role in catalysis/binding. Inthe present study we have identified elementary steps and intermediates in the reaction pathway of plasmidDNA cleavage by MunI and elucidated the effect of pH and Mg2+ ions on the individual steps of theDNA cleavage reaction. The kinetic analysis indicated that the multiple-turnover rate of plasmid cleavageby MunI is limited by product release throughout the pH range 6.0−9.3. Quenched-flow experimentsrevealed that open circle DNA is an obligatory intermediate in the reaction pathway. Under optimal reactionconditions, open circle DNA remains bound to the MunI; however it is released into the solution at low[MgCl2]. Rate constants for the phoshodiester bond hydrolysis of the first (k1) and second (k2) strand ofplasmid DNA at pH 7.0 and 10 mM MgCl2 more than 100-fold exceed the kcat value which is limitedby product dissociation. The analysis of the pH and [Mg2+] dependences of k1 and k2 revealed that bothH+ and Mg2+ ions compete for the binding to the same residue at the active site of MunI. Thus, thedecreased rate of phosphodiester hydrolysis by MunI at pH < 7.0 may be due to the reduction of affinityfor the Mg2+ binding at the active site. Kinetic analysis of DNA cleavage by MunI yielded estimates forthe association−dissociation rate constants of enzyme−substrate complex and demonstrated the decreasedstability of the MunI−DNA complex at pH values above 8.0.
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