Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism.
DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in KmDNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Double-reciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dI.dC-dI.dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M. HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.
Flynn
J
J
Department of Chemistry, Program in Biochemistry and Molecular Biology, University of California, Santa Barbara 93106, USA.
Reich
N
N
D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019175:DNA Methylation; D007553:Isotope Labeling; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D011089:Polydeoxyribonucleotides; C031156:poly d(I-C); D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D004248:DNA (Cytosine-5-)-Methyltransferases