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 [generating N(6)-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.
Malygin
E G
EG
Institute of Molecular Biology, State Research Center of Virology and Biotechnology 'Vector', Novosibirsk 633159, Russia.
Lindstrom
W M
WM
Schlagman
S L
SL
Hattman
S
S
Reich
N O
NO
D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D012436:S-Adenosylmethionine; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499297:dam protein, Bacteriophage T4