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KMID : 0380219960290020099
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Journal of Biochemistry and Molecular Biology
1996 Volume.29 No. 2 p.99 ~ p.104
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Site-Directed Mutagenesis Studies with Restriction Endonuclease EcoRV to Identify the Role of Ile91 in Recognition and Catalysis
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Moon Byung-Jo
Vipond I. Barry
Halford Stephen E.
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Abstract
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Site-directed substitutions were made to change the Ile91 of restriction endonuclease EcoRV to either Val, Ala or Gly to identify the role of Ile91 in recognition and catalysis, since substitution of Ile91 with Leu afforded dramatic effects on the activity and properties of restriction endonuclease EcoRV. These changes alter the size of the hydrophobic side chain at position 91 and thus might have revealed the reason for the altered phenotype of Ile91Leu. However, the properties of Ile91Val and Ile91Ala mutants were much like wild type EcoRV, in both activity and metal ion preference. Ile91Gly had very little activity with either Mg2+ or Mn2+ as cofactors. To try to understand the unusual Mn2+ profile of the Ile91Leu mutant, two double mutants, Ile91Leu;Asp90Asn and Ile91Leu;Glu45Met were created. Both double mutants were seriously disabled by the second amino acid change. Ile91Leu;Glu45Met had some residual activity in the Mn2+ reaction buffer, whereas the Ile91Leu;Asp90Asn displayed no detectable activity.
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KEYWORD
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restriction endonuclease EcoRV, site-directed mutagenesis
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