Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase

Bheemanaik, Shivakumar ; Chandrashekaran, Siddamadappa ; Nagaraja, Valakunja ; Rao, Desirazu Narasimha (2003) Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase Journal of Biological Chemistry, 278 (10). pp. 7863-7874. ISSN 0021-9258

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Official URL: http://www.jbc.org/content/278/10/7863.full

Related URL: http://dx.doi.org/10.1074/jbc.M211458200

Abstract

KpnI DNA-(N6-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 N6 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 thatS-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.

Item Type:Article
Source:Copyright of this article belongs to American Society for Biochemistry and Molecular Biology.
ID Code:26951
Deposited On:08 Dec 2010 12:57
Last Modified:17 May 2016 10:14

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