title: Kinetic mechanism of the EcoRI DNA methyltransferase.
abstract: We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.
authors.affiliation: Chemistry Department, University of California, Santa Barbara 93106.
authors.forename: N O
authors.initials: NO
authors.lastname: Reich
authors.forename: N
authors.initials: N
authors.lastname: Mashhoon
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D012436:S-Adenosylmethionine; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)