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The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease

1989; Elsevier BV; Volume: 264; Issue: 20 Linguagem: Inglês

10.1016/s0021-9258(18)80138-7

1083-351X

David J. Wright, Kendall W. King, Paul Modrich,

Bacterial Genetics and Biotechnology

1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition.We have substituted Gln or Asp for Glu-11 1 by oligonucleotide-directed mutagenesis.First and second strand cleavage rate constants are reduced by a factor of more than lo4 by the Gln-111 substitution.However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI* sites by wild type endonuclease to a similar degree.The specific affinity of Gln-11 1 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation.In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-11 1 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme.These results suggest that a negative charge on residue 11 1 is required for strand cleavage and are consistent with participation of Glu-11 1 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.