DNA (cytosine-N4-)- and -(adenine-N6-)-methyltransferases have different kinetic mechanisms but the same reaction route. A comparison of M.BamHI and T4 Dam.
We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-l-homocysteine, AdoHcy) was Ado-Met DNA DNA(Me) AdoHcy. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N(6)-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.
Malygin
Ernst G
EG
Institute of Molecular Biology, State Research Center of Virology and Biotechnology "Vector," Koltsovo, Novosibirsk Region, 630559 Russia.
Zinoviev
Victor V
VV
Evdokimov
Alexey A
AA
Lindstrom
William M
WM
Reich
Norbert O
NO
Hattman
Stanley
S
D002384:Catalysis; D019175:DNA Methylation; D015245:Deoxyribonuclease BamHI / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014764:Viral Proteins
D014764:Viral Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499297:dam protein, Bacteriophage T4; D015245:Deoxyribonuclease BamHI