[Single turnover kinetics of phage T4 DNA-(N6-adenine)methyltransferase]

Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition 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 steady-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. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical 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 (k1 = 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 (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.