THE RESTRICTION ENDONUCLEASE ECO RI: ITS INTRINSIC METAL CENTER AND SPECIFIC INTERACTIONS WITH A DNA RECOGNITION SITE (PROTEIN-NUCLEIC ACID, ZINC METALLOENZYMES, INTERACTIONS CHIRAL METAL COMPLEXES, DNA-BINDING PROTEINS)

Protein-nucleic acid interactions play an important role in life processes such as replication, transcription and regulation of gene expression. In an attempt to characterize the nature of these interactions, the restriction enzyme Eco RI was examined for its metal content. This site-specific DNA cleaving enzyme contains one tightly bound zinc atom per subunit. Removal of the zinc ion results in loss of endonuclease activity indicating that the metal is an integral component of the protein. Addition of the zinc ion back to the inactive protein restores the catalytic activity. The replacement of the native zinc ion with cobalt produces active endonuclease having the same site-specificity for DNA. The enzyme containing increasing amounts of substituted cobalt (0-80%), shows a gradual increase in its apparent dissociation constant (4.3-36 nM) for the substrate Col E(,1)DNA. The maximal velocity of the reaction is increased from 0.63 for the native enzyme to 3.3 nM/minute for the cobalt enzyme. These kinetic studies indicate the direct participation of the metal ion in enzyme activity. Chelators having high affinity for zinc inhibit the enzyme. Some of these ligands are competitive inhibitors implying that they inhibit the reaction by coordinating to zinc ion. This observation again suggests that the metal is necessary for Eco RI activity. The enzyme-bound zinc ion may also be replaced by cobalt ion in vivo, indicating that the metal ion is intrinsic to the protein. The visible absorption spectrum of Co-Eco RI resembles that of Co-Zn RNA polymerase, showing two intense peaks between 375-475 nm and a much weaker peak at 590 nm. The former bands were perturbed in the presence of substrate DNA implying that the metal may be required for substrate binding. The structure of the DNA site in the bound protein-SNA complex was also probed using chiral metal complexes which are DNA conformational probes. Site-specific cleavage reaction with the metal complexes indicated that a structural change of DNA occurred near the enzyme-bound site. Thus the results illustrate in solution the alteration in DNA conformation as a result of the site-specific association of Eco RI.