Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease.
The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.
Hancox
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Department of Biochemistry, University of Bristol, U.K.
Halford
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D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D007930:Leucine; D008239:Lysine; D008274:Magnesium / Q000494:pharmacology; D016296:Mutagenesis; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D005973:Glutamine; D018698:Glutamic Acid; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D007930:Leucine; D008274:Magnesium; D008239:Lysine; D002118:Calcium