THE MECHANISM OF DNA CLEAVAGE BY THE TYPE-II RESTRICTION ENZYME ECORV- ASP36 IS NOT DIRECTLY INVOLVED IN DNA CLEAVAGE BUT SERVES TO COUPLEINDIRECT READOUT TO CATALYSIS

Three different mechanisms have been proposed to describe DNA cleavage by the type II restriction endonuclease EcoRV, which differ in the nu mber and function of metal ions directly involved in catalysis and the different roles assigned to amino acid residues in the active sites a nd a phosphate group of the substrate. There are only four acidic amin o acid residues close to the scissile bond: the essential Asp74 and As p9O, the non-essential Glu45, and Asp36, We show here that Asp36 can b e exchanged for alanine, with only minor effects on the cleavage rate of the nearby phosphodiester bond, excluding that Asp36 could be direc tly involved in catalysis, Hence, the two versions of the two-metal-io n mechanism are not compatible with the experimental data, because too few ligands for two metal ions are present near the active site of Ec oRV, Our result, thus, supports the one-metal-ion mechanism for EcoRV, We suggest that Asp36 has an allosteric effect by which specific cont acts between one strand of the DNA and one subunit of the enzyme trigg er the activation of one catalytic center, Given the similar structure s of the active sites of EcoRV, EcoRI, BamHI, Pvull and Fokl, as well as the occurrence of a characteristic catalytic motif in several other restriction enzymes, we conclude that these enzymes most likely share a similar mechanism of DNA cleavage, whose characteristic feature is the involvement of only one Mg2+ ion in catalysis.