Two Identical Subunits of the EcoRI Restriction Endonuclease Co‐operate in the Binding and Cleavage of the Palindromic Substrate

Abstract

The cleavage of radioactively labelled double‐stranded d(G‐G‐A‐A‐T‐T‐C‐C) was studied in single turnover experiments with substrate and enzyme both being in the micromolar range. The reaction rate was found to increase with enzyme concentration until a ratio of one tetrameric enzyme to two double‐stranded substrates was reached, further increase of the enzyme concentration then leads to a sharp decline of the reaction rate. These finding are interpreted in the following manner. (a) Two subunits of the EcoRI endonuclease co‐operate in binding and possibly also in cleaving the palindromic substrate. (b) The enzymatic action of the EcoRI endonuclease is inhibited by excess enzyme, possibly due to unspecific binding of the enzyme‐substrate complex. The sell‐inhibition of EcoRI endonuclease has also been observed with macromolecular substrates. Copyright © 1982, Wiley Blackwell. All rights reserved