Purification and biochemical characterization of the Ecal DNA methyltransferase

László SZILÁK, Pál VENETIANER, Antal KISS

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Abstract

The EcaI GGTNACC‐specific DNA‐adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The Km was 170 nM for DNA and 1.8 μM for the methyl donor S‐adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki= 3.5 nM). The other product of the DNA‐methylation reaction, S‐adenosylhomocysteine was found to be a competitive inhibitor with respect to S‐adenosylmethionine (Ki= 2.7 μM). The S‐adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki= 3.5 nM) of the DNA methyltransferase reaction.

Original languageEnglish
Pages (from-to)391-397
Number of pages7
JournalEuropean Journal of Biochemistry
Volume209
Issue number1
DOIs
Publication statusPublished - Oct 1992

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ASJC Scopus subject areas

  • Biochemistry

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