Pre-steady state kinetics of bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase: Interaction with native (GATC) or modified sites

Abstract

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 N6-position of adenine [generating N6-methyladenine and S-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant K(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s-1, respectively) 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 nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.