Purification and biochemical characterization of the EcaI DNA methyltransferase.
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 degrees 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 microM 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 microM). The S-adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.
Szilák
L
L
Institute of Biochemistry, Hungarian Academy of Sciences, Szeged.
Venetianer
P
P
Kiss
A
A
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D004247:DNA; D012435:S-Adenosylhomocysteine; C063674:DNA modification methylase EcaI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000241:Adenosine; C006235:sinefungin