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KMID : 0380219960290010017
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Journal of Biochemistry and Molecular Biology
1996 Volume.29 No. 1 p.17 ~ p.21
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Site-Directed Mutagenesis of Ile91 of Restriction Endonuclease EcoRV: Dramatic Consequences on the Activity and the Properties of the Enzyme
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Moon Byung-Jo
Vipond I. Barry
Halford Stephen E.
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Abstract
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Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers Mn2+ to Mn2+ as the cofactor. In Mn2+ buffer the mutant is as active as the wild type enzyme in Mn2+ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.
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KEYWORD
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restriction endonuclease EcoRV, site-directed mutagenesis
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