EcoRV restriction endonuclease: communication between DNA recognition and catalysis.
A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.
Vermote
C L
CL
Department of Biochemistry, University of Bristol, U.K.
Vipond
I B
IB
Halford
S E
SE
D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003460:Crystallization; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D015394:Molecular Structure; D016296:Mutagenesis; D010957:Plasmids; D014169:Transformation, Bacterial
D008345:Manganese; D001216:Asparagine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium