An Isoleucine to Leucine Mutation that Switches the Cofactor Requirement of the EcoRV Restriction Endonuclease from Magnesium to Manganese

• Published in: Biochemistry (Easton) 1996-02, Vol.35 (6), p.1712-1721
• Main Authors: Vipond, I. Barry, Moon, Byung-Jo, Halford, Stephen E
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites - genetics; Deoxyribonucleases, Type II Site-Specific - chemistry; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Isoleucine - chemistry; Kinetics; Leucine - chemistry; Magnesium - metabolism; Manganese - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Point Mutation; Protein Conformation; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9523926

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme:  the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile→Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV.