Kinetic characterization of the EcaI methyltransferase.
A kinetic analysis of the EcaI adenine-N6-specific methyltransferase (MTase) is presented. The enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the adenine of the GGTNACC sequence with a random rapid-equilibrium mechanism. Experiments with a synthetic, 14-bp DNA substrate suggest that recognition of the specific site of DNA occurs after the binding of AdoMet. Proton concentration does not affect the dissociation constant of AdoMet while Vm and the dissociation constant of DNA show a maximum around pH 8. Increasing the amount of S-adenosyl-L-homocysteine decreases the inhibitory effect of methylated DNA which proves the active role of AdoMet in site recognition. Experiments with hemimethylated DNA show that the methylase binds the double-stranded DNA asymmetrically.
Szilák
L
L
Institute of Biochemistry, Hungarian Academy of Sciences, Szeged.
Dér
A
A
Deák
F
F
Venetianer
P
P
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D012436:S-Adenosylmethionine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C032823:GGTNACC-specific type II deoxyribonucleases