title: Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase.
abstract: KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Shivakumara
authors.initials: S
authors.lastname: Bheemanaik
authors.forename: Siddamadappa
authors.initials: S
authors.lastname: Chandrashekaran
authors.forename: Valakunja
authors.initials: V
authors.lastname: Nagaraja
authors.forename: Desirazu N
authors.initials: DN
authors.lastname: Rao
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D017931:DNA Primers; D019281:Dimerization; D007700:Kinetics; D008745:Methylation; D008970:Molecular Weight; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; C072049:DNA modification methylase KpnI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)