DNA-(N4-cytosine)-Methyltransferase from Bacillus amyloliquefaciens: Kinetic and substrate-binding properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amylol iquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindr omic recognition site GGATCC was studied by methods of steady-state and pre -steady-state kinetics of the methyl group transfer, gel retardation, and c rosslinking of the enzyme subunits with glutaraldehyde. In steady-state con ditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer s ubstrate. A linear dependence was observed for the reaction rate on the enz yme concentration and a Michaelis dependence of the reaction rate on the co ncentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl g roup donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme conc entration being substantially lower than the concentrations of substrates. The k(cat) values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s(-1). The K-M values for the duplex and SAM are 0.35 and 1.6 muM, respectively. An analysis of single tu rnover kinetics (at limiting concentration of the 20-mer duplex) revealed t he following characteristics of the BamHI MTase-dependent methylation of DN A. The value of rate constant of the DNA methylation step at the enzyme sat urating concentration is on average 0.085 s(-1), which is only 1.6 times hi gher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the or der of enzyme preincubation with SAM and DNA, both curves for the single tu rnover methylation are comparable. These results are consistent with the mo del of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI M Tase in the steady-state reaction are the data on the enzyme binding with D NA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was sh own that the enzyme in a free state exists as a dimer. Introduction of subs toichiometric amounts of DNA into the reaction mixture results in pronounce d multimerization of the enzyme. However, addition of SAM in saturating con centration at an excess of the oligonucleotide duplex over BamHI MTase conv erts most of the enzyme into a monomeric state.