Pre-steady state kinetics of bacteriophage T4 Dam DNA-[N-6-adenine] methyltransferase: interaction with native (GATC) or modified sites

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to the N-6-position of adenine [generatin g N-6-methyladenine and S-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady sta te kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectiv ely) was at least 20-fold larger than the overall reaction rate constant k( cat) (0.023 s(-1)). This indicates that the release of products is the rate -limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleos ide flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes a re less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route o f reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.