Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli

Rao, Desirazu N. ; Page, Malcolm G. P. ; Bickle, Thomas A. (1989) Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli Journal of Molecular Biology, 209 (4). pp. 599-606. ISSN 0022-2836

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/002228...

Related URL: http://dx.doi.org/10.1016/0022-2836(89)90597-4

Abstract

The EcoP15 modification methylase gene from the p15B plasmid of Escherichia coli 15T has been cloned and expressed at high levels in a plasmid vector system. We have purified the enzyme to near homogeneity in large amounts and have studied some of its enzymatic properties. Initial rates of methyl transfer are first order in methylase concentration and, with pUC19 DNA as substrate, the reaction proceeds by a random mechanism in which either DNA or S-adenosylmethionine can bind to the free enzyme. After methyltransfer for DNA, the methylated DNA and S-adenosylhomocysteine appear to dissociate in random order. As expected in such a mechanism, S-adenosylhomocysteine is a non-competitive inhibitor with respect to both S-adenosylmethionine and DNA. DNA-dependent substrate inhibition by S-adenosylmethionine at concentrations not much above its KM suggests that release of methylated DNA may be the rate-limiting step. This suggestion is strengthened by the fact that a mutant of the closely related EcoP1 does not show such substrate inhibition.

Item Type:Article
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