Single turnover kinetics of methylation by T4 DNA-(N6-adenine)-methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase was studied with a var iety of synthetic oligonucleotide substrates containing the native recognit ion site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the stea dy-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Up on collision of a T4 MTase monomer with an oligonucleotide duplex, an asymm etrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k(1 ) = 0.21 s(-1)). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k(2) = 0.023 s(-1) for duplex with the native site), the dimeric T4 MTase switches over to th e second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is m uch higher than the overall rate of the T4 MTase-catalyzed steady-state rea ction, although this difference is considerably less than that shown for Ec oRI MTase. Base substitutions and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is di srupted, the proportion of the initial productive enzyme-substrate complexe s is usually sharply reduced. The flipping of the adenosine residue to be m odified in the recognition site upon interaction with the enzyme, revealed by fluorescence titration, supports the existing notions about the involvem ent of such a DNA deformation in reactions catalyzed by various DNA-MTases.